Secondary alcohol dehydrogenase enzyme and use thereof

ABSTRACT

Disclosed is a newly-discovered and isolated enzyme which is a nicotinamide adenine dinucleotide (NAD + )-linked C 3  -C 6  secondary alcohol-specific dehydrogenase. This enzyme, in the presence of oxygen and NAD, is capable of converting C 3  -C 6  secondary alcohols to the corresponding methyl ketones. The purified enzyme has a molecular weight of 95,000 ± 3,000 dalton as determined by acrylamide gel electrophoresis and contains two zinc atoms per molecule of protein. Primary alcohols are not oxidized by this enzyme.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. applications Ser.Nos. 896,475 and 896,476, filed Apr. 14, 1978, both now abandoned. Thisapplication is related to U.S. application Ser. No. 896,467; filed Apr.14, 1978, now abandoned.

FIELD OF THE INVENTION

The present invention relates to newly discovered and isolatedmethylotrophic microorganism strains and their natural and/or artificialmutants which grow well under aerobic conditions in a culture medium inthe presence of methane as the major carbon and energy source. Themethane-grown microbial cells are high in protein and are useful asfeedstuffs. The methane-grown microbial cells or enzyme preparationsthereof are also useful in converting oxidizable substrates to oxidizedproducts, e.g., C₁ -C₆ alkanes to alcohols, C₃ -C₆ alkanes to thecorresponding C₃ -C₆ sec. alcohols and methyl ketones, C₃ -C₆ sec.alcohols to the corresponding methyl ketones, cyclic hydrocarbons tocyclic hydrocarbyl alcohols (e.g., cyclohexane to cyclohexanol), C₂ -C₄alkenes selected from the group consisting of ethylene, propylene,butene-1 and butadiene to the corresponding 1,2-epoxides, styrene tostyrene oxide, etc.

BACKGROUND OF THE INVENTION

Methane is one of the most inexpensive carbon sources for microbialgrowth. It is known that there are many microorganisms capable ofgrowing on a culture medium in the presence of methane as the principlecarbon source. However, not all of these microorganisms share goodgrowth characteristics. It is also known that methane-grownmicroorganisms can be used to convert methane to methanol under aerobicconditions.

These methane-utilizing microorganisms are generally known as"methylotrophs". The classification system for methylotrophs proposed byR. Whittenbury et al. (J. of Gen. Microbiology, 61, 205-218 (1970)) isthe most widely recognized. In their system, the morphologicalcharacteristics of methane-oxidizing bacteria are divided into fivegroups: Methylosinus, Methylocystis, Methylomonas, Methylobacter andMethylococcus.

Recently, Patt, Cole and Hanson (International J. SystematicBacteriology, 26, (2) 226-229 (1976)) disclosed that methylotrophicbacteria are those bacteria that can grow non-autotrophically usingcarbon compounds containing one or more carbon atoms but containing nocarbon-carbon bonds. Patt et al. have proposed that methylotrophs shouldbe considered "obligate" if they are capable of utilizing only carboncompounds containing no carbon-carbon bonds (e.g., methane, methanol,dimethylether, methylamines, etc.) as the sole sources of carbon andenergy whereas "facultative" methylotrophs are those organisms that canuse both compounds containing no carbon-carbon bonds as well ascompounds having carbon-carbon bonds as the sources of carbon andenergy. In their paper, Patt et al. disclosed a methane-oxidizingbacterium, which they identified as Methylobacterium organophilum spnov. (ATCC 27,886). This bacterium presumably differs from allpreviously described genera and species of methane-oxidizing bacteriabecause of its ability to utilize a variety of organic substrates withcarbon-carbon bonds as sources of carbon and energy.

DESCRIPTION OF THE PRIOR ART

Hutchinson, Whittenbury and Dalton (J. Theor. Biol., 58, 325-335 (1976)"A Possible Role of Free Radicals in the Oxidation of Methane byMethylococcus capsulatus") and Colby and Dalton (J. Biochem., 157,495-497 (1976) "Some Properties of a Soluble Methane Mono-Oxygenase FromMethylococcus capsulatus Strain Bath") reported that ethylene isoxidized by the soluble methane mono-oxygenase from Methylococcuscapsulatus Strain Bath. The latter investigators reported that the"particulate membrane preparations" of Methylococcus capsulatus StrainBath did not have methane-oxygenase activity as determined by thebromomethane disappearance test.

Cerniglia, Blevins and Perry, (Applied and Environmental Microbiology,32, (6) 764-768 (1976) "Microbial Oxidation and Assimilation ofPropylene") described the oxidation of propylene by microorganisms tothe corresponding alcohols and carboxylic acids.

Most recently, Colby, Stirling and Dalton, (J. Biochem., 165, 395-402(August, 1977)) "The Soluble Methane Mono-Oxygenase of Methylococcuscapsulatus (Bath) Its Ability to Oxygenate n-Alkanes, n-Alkenes, Ethers,and Alicyclic Aromatic and Heterocyclic Compounds") disclosed that thesoluble fraction of Methylococcus capsulatus Strain Bath is a verynon-specific oxygenase in that it oxidizes alkanes to alcohols, alkenesto 1,2-epoxides, dimethylether to ethanol and ethanal, styrene tostyrene epoxide and pyridine to pyridine N-oxide.

On the basis of ¹⁸ O₂ incorporation from ¹⁸ O₂ into the cellularconstituents of Pseudomonas methanica Leadbetter and Foster (Nature,184: 1428-1429 (1959) "Incorporation of Molecular Oxygen in BacterialCells Utilizing Hydrocarbons For Growth") suggested that the initialoxidative attack on methane involves an oxygenase. Higgins and Quayle(J. Biochem., 118: 201-208 (1970) "Oxygenation of Methane byMethane-Grown Pseudomonas methanica and Methanomonas methanooxidans")isolated CH₃ ¹⁸ OH as the product of methane oxidation when suspensionsof Pseudomonas methanica or Methanomonas methanooxidans were allowed tooxidize methane in ¹⁸ O₂ -enriched atmospheres. The subsequentobservation of methane-stimulated NADH oxidation catalyzed by extractsof Methylococcus capsulatus by Ribbons (J. Bacteriol., 122: 1351-1363(1975) "Oxidation of C₁ -Compounds by Particulate Fractions FromMethylococcus capsulatus: Distribution and Properties ofMethane-Dependent Reduced Nicotinamide Adenine Dinucleotide Oxidase(methane hydroxylase)") and Ribbons and Michalover, FEBS Lett. 11: 41-44(1970) "Methane Oxidation by Cell-Free Extracts of Methylococcuscapsulatus" or Methylomonas Methanica Ferenci (FEBS Lett. 41: 94-98(1974) "Carbon Monoxide-Stimulated Respiration in Methane-UtilizingBacteria") suggested that the enzyme responsible for this oxygenation isa monooxygenase. These workers relied on indirect enzyme assays,measuring methane-stimulated NADH disappearance spectrophotometricallyor methane-stimulated O₂ disappearance polarographically. Recently,methane monooxygenase systems were partially purified from Methylosinustrichosporium OB3b (Tonge, Harrison and Higgins, J. Biochem., 161:333-344 (1977) "Purification and Properties of the Methane MonooxygenaseEnzyme System From Methylosinus trichosporium OB3b"); and Tonge,Harrison, Knowles and Higgins, FEBS Lett., 58: 293-299 (1975)"Properties and Partial Purification of the Methane-Oxidizing EnzymeSystem From Methylosinus trichosporium") and Methylococcus capsulatus(Bath) (Colby and Dalton, J. Biochem., 171: 461-468 (1978) "Resolutionof the Methane Mono-Oxygenase of Methylococcus capsulatus (Bath) IntoThree Components" and Colby, Stirling and Dalton, J. Biochem., 165:395-402 (1977) "The Soluble Methane Mono-Oxygenase of Methylococcuscapsulatus (Bath), "Its Ability to Oxygenate n-Alkanes, n-Alkenes,Ethers, and Alicyclic, Aromatic and Heterocyclic Compounds").

The microbiological formation of methyl ketones in mammals, bacteria andfungi is well known. However, the ketone is formed by decarboxylation ofa beta-keto acid and has, therefore, one less carbon atom than theprecursor. On the other hand, bacterial formation of methyl ketones fromn-alkanes, demonstrated first by Leadbetter and Foster (Arch.Mikrobiol., 35: 92-104 (1960)) represents a unique alpha-oxidation, withno change in the carbon skeleton. However, in this letter report it wasstated that the ketone formation was by co-oxidation in the presence ofthe growth substrate and indicated that no activity was found with theresting cells.

Phenazine methosulfate (PMS)-dependent methanol dehydrogenase has beenextensively reported from many methylotrophic bacteria. This enzymeoxidizes primary alcohols from C₁ to C₁₀ but does not oxidize secondaryalcohols. Nicotinamide ademine dinucleotide (NAD)-dependent alcoholdehydrogenases have been reported from liver and from yeast. Thesealcohol dehydrogenases oxidize primary alcohols and acetaldehyde, buthave no activity on methanol. In addition, the alcohol dehydrogenasesfrom yeast and liver also oxidize some secondary alcohols at a very lowrate (<1% of their ethanol activity). NAD(P)-dependent alcoholdehydrogenases were also reported in Pseudomonas, E. coli andLeuconostoc. However, these enzymes were active only toward long-chainprimary alcohols or hydroxy fatty acids. Recently, an NAD-linkedmethanol oxidizing enzyme was reported in a crude extract from yeast(Mehta, R. J., J. Bacteriol., 124, 1165-1167 (1975). To our knowledgethere is no report in the literature of a secondary alcohol-specificalcohol dehydrogenase (SADH) enzyme.

Since Ogata et al. (J. Ferm. Technol., 48: 389-396 (1970)) firstreported the assimilation of methanol by a yeast, manymethanol-utilizing strains have been isolated from natural sources orfound in stock culture collections. Interest in the cultivation ofmicroorganisms on cheap and abundantly available compounds, such asmethanol has increased greatly as a result of the potential importanceof microbial protein as a food or fodder material. The production ofsingle-cell protein (SCP) from methanol-grown yeasts have been discussedin several publications. Oxidation of methanol and other primaryalcohols in yeasts has been shown to be catalyzed by an alcohol oxidase.Alcohol oxidase contained flavin adenine dinucleotide (FAD) as aprosthetic group. Secondary alcohols were not oxidized by this alcoholoxidase.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

It has now been discovered that certain newly discovered and isolatedmethylotrophic microorganism strains and their natural and/or artificialmutants thereof grow well under aerobic conditions in a culture mediumin the presence of methane as the major carbon and energy source. Themethane-grown microbial cells possess a high content of protein and canbe utilized as such as feedstuffs. The methane-grown microbial cells orenzyme preparations thereof are also useful in converting oxidizablesubstrates to oxidation products, e.g., C₁ -C₆ alkanes to alcohols,particularly methane to methanol, C₃ -C₆ alkanes to the correspondingsecondary alcohols and methyl ketones, C₃ -C₆ sec. alcohols to thecorresponding methyl ketones, cyclic hydrocarbons to cyclic hydrocarbylalcohols (e.g., cyclohexane to cyclohexanol), C₂ -C₄ alkenes selectedfrom the group consisting of ethylene, propylene, butene-1, andbutadiene to 1,2-epoxides, styrene to styrene oxide, etc.

It has also been discovered that these newly discovered and isolatedmethylotrophic microorganism strains, including new yeast strains, maybe aerobically grown on a plurality of methyl radical donatingcarbon-containing compounds, such as methanol, methylamine, methylformate, methyl carbonate, dimethyl ether, etc., to produce microbialcells or enzyme preparations capable of aerobically converting C₃ -C₆linear secondary alcohols to the corresponding methyl ketones.

As an additional discovery we have identified a nicotinamide adeninedinucleotide (NAD)-dependent secondary alcohol dehydrogenase incell-free extracts of various hydrocarbon-utilizing microbes, includingbacteria and yeast. This enzyme is also found in cells grown onmethanol. It specifically and stoichiometrically oxidizes C₃ -C₆secondary alcohols to their corresponding methyl ketones. This enzymehas been purified 2600 fold and shows a single protein band onacrylamide gel electrophoresis. It has a molecular weight of 95,000dalton. The bacterial SADH consists of two subunits of 48,000 dalton andto atoms of zinc per molecule of enzyme protein. It oxidizes secondaryalcohols, notably 2-propanol and 2-butanol. Primary alcohols are notoxidized by SADH.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The term "microorganism" is used herein in its broadest sense to includenot only bacteria, but also yeasts, filamentous fungi, actinomycetes andprotozoa. Preferably, the microorganisms will include bacteria, and morepreferably the bacteria capable of oxidizing methane and methyl-radicaldonating carbon-containing compounds.

The term "enzyme preparation" is used to refer to any composition ofmatter that exhibits the desired oxygenase or dehydrogenase enzymaticactivity. The term is used to refer, for example, to live whole cells,dried cells, cell extracts and refined and concentrated preparationsderived from the cells, especially purified secondary alcoholdehydrogenase and its NAD⁺ cofactor and metal requirement. Enzymepreparations may be either in dry or liquid form. The term also includesthe immobilized form of the enzyme, e.g., the whole cells of the methaneor methyl-radical-grown microorganisms or enzyme extracts immobilized orbound to an insoluble matrix by covalent chemical linkages, absorptionand entrapment of the enzyme within a gel lattice having pores largeenough to allow the molecules of the substrate and of the product topass freely, but small enough to retain the enzyme. The term "enzymepreparation" also includes enzymes retained within hollow fibermembranes, e.g., as disclosed by Rony, Biotechnology and Bioengineering(1971).

The term "particulate fraction" refers to the enzyme activity in theprecipitated or sedimented material when the supernatant aftercentrifuging broken cells at 10,000×g. for 30 minutes is centrifuged for1 hour at 10,000×g. or greater.

The term "increasing the oxidative state of an oxidizable organicsubstrate" is meant to include incorporating oxygen in an organiccompound, such as in epoxidizing olefins and converting alkanes toalcohols or ketones or increasing the oxidative state ofoxygen-containing compounds such as converting alcohols to aldehydes andketones (i.e., a dehydrogenating reaction). Specifically preferredprocesses where the methane or methyl-radical-grown microbial cells ortheir enzyme preparations are used to increase the oxidative state of anoxidizable organic substrate include: converting C₂ -C₄ alkenes selectedfrom the group consisting of ethylene, propylene, butene-1 and butadieneto 1,2-epoxides; converting C₁ -C₆ alkanes to corresponding alkanols;converting C₃ -C₆ sec. alcohols to the corresponding methyl ketones. Inthis regard, microbial cells or enzyme preparations of methane-grownMethylobacter vinelandii M5Y NRRL B-11,218 and mutants thereof convertscyclohexane to cyclohexanol.

The classification system of methane-oxidizing bacteria proposed by R.Whittenbury, K. C. Phillips and J. F. Wilkinson [J. Gen. Microbiology,61, 205-218 (1970) (hereinafter Whittenbury et al.)] is the most widelyrecognized system used today. In this system of classification, based onmorphological characteristics methane-utilizing bacteria are dividedinto five groups. They are: Methylosinus, Methylocystis, Methylomonas,Methylobacter and Methylococcus. Bacteria of these five groups reportedby Whittenbury et al. utilize methane, dimethyl ether, and methanol forgrowth energy and they were all reported as strictly aerobic andgram-negative.

As one embodiment of the present invention, we have discovered andisolated several new strains which grow well on a culture medium in thepresence of oxygen and methane and methyl-radical donating compoundssuch as methanol, methylamine, methyl formate, methyl carbonate,dimethyl ether, etc. These newly discovered and isolated strains ofmethylotrophic microorganisms are capable of producing microbial cellsuseful as feedstuffs when cultured under aerobic conditions in a liquidgrowth medium comprising assimilable sources of nitrogen and essentialmineral salts in the presence of methane gas or the above-mentionedmethyl-radical donating carbon-containing compounds as the major carbonand energy source.

As another embodiment of the invention there is provided biologicallypure isolates and mutants thereof of a plurality of newly discovered andisolated methane-utilizing microorganism strains. These biologicallypure isolates (described in more detail below) are capable of producingmicrobial cells when cultivated in an aerobic nutrient medium containingmethane or the above-mentioned methyl-radical donating carbon-containingcompounds as the major carbon and energy source.

As still another embodiment of the invention there is provided a processfor increasing the oxidative state of an oxidizable organic substratewhich comprises contacting, under aerobic conditions, in a mediumcomprising assimilable sources of nitrogen and essential mineral salts,microbial cells or an enzyme preparation thereof and an organicsubstrate until the oxidative state of at least a portion of saidorganic substrate is increased, wherein said microbial cells have beencultured, under aerobic conditions in a liquid growth medium comprisingassimilable sources of nitrogen and essential mineral salts in thepresence of methane gas as the major carbon and energy source whereinthe microbial cells are derived from the newly discovered and isolatedmethane-utilizing strains of the invention (described below).

A particularly preferred embodiment of the invention includes a processfor producing propylene oxide from propylene by contacting propyleneunder aerobic conditions with microbial cells or enzyme preparationthereof wherein the microbial cells are derived from the newlydiscovered and isolated methane-utilizing strains of the presentinvention as described below and which have been previously grown underaerobic conditions in the presence of methane.

Another particular preferred embodiment of the invention includes aprocess for converting C₃ -C₆ linear secondary alcohols to thecorresponding methyl ketones by contacting a C₃ -C₆ linear secondaryalcohol under aerobic conditions with microbial cells or enzymepreparations thereof (including cell extracts or purified SADH and NAD⁺)wherein the microbial cells are derived from the newly discovered andisolated methane or methyl-radical utilizing strains of the presentinvention as described below and which have been previously grown underaerobic conditions in the presence of methane or a methyl-radicaldonating carbon-containing compound such as methanol, methylamine,methyl formate, methyl carbonate, dimethyl ether, etc., most preferablymethane or methanol.

The instant invention includes the following features:

The isolates of methane-utilizing microbes of the invention includeobligate (Type I and Type II) and facultative bacteria as well as newmethanol utilizing yeasts.

In addition to their ability to oxidize methane to methanol, restingcell-suspensions of several distinct types of methane-grown bacteria(e.g., Type I, obligate; Type II, obligate; and facultative)oxidize C₂-C₄ n-alkene and butadiene to their corresponding 1,2-epoxides.

The product 1,2-epoxides are not further metabolized and accumulateextracellularly.

Methanol-grown cells do not have either the epoxidation or thehydroxylation activities. Among the substrate gaseous alkenes, propyleneis oxidized at the highest rate.

Methane inhibits the epoxidation of propylene.

The stoichiometry of the consumption of propylene and oxygen, and theproduction of propylene oxide is 1:1:1.

Results from inhibition studies indicate that the same monooxygenasesystem catalyzes both the hydroxylation and the epoxidation reactions.

Both the hydroxylation and epoxidation activities are located in thecell-free (enzyme extract) particulate fraction precipitated orsedimented between 10,000×g. and 80,000×g. centrifugation for 1 hour.

Cell-free particulate fractions from the obligate and facultativemethylotroph microorganisms catalyze the hydroxylation of methane tomethanol and the epoxidation of C₂ -C₄ n-alkenes and dienes (e.g.,ethylene, propylene, 1-butene and butadiene) in the presence of oxygenand reduced nicotinamide adenine dinucleotide (NADH) and thehydroxylation of C₁ -C₄ n-alkanes (e.g., methane, ethane, propane andbutane).

The hydroxylation and epoxidation activities of the methane-grownmethylotrophs are lost simultaneously during storage and are stronglyinhibited by various metal-binding agents.

The stoichiometry for the consumption of substrate (propylene ormethane), oxygen, NADH, and product formation was found to beapproximately 1:1:1:1.

Resting-cell suspensions of the new C₁ -utilizing microbes oxidize(dehydrogenate) C₃ -C₆ secondary alcohols to their corresponding methylketones. The product methyl ketones accumulate extracellularly. Amongthe secondary alcohols, 2-butanol was oxidized at the highest rate.

Succinate-grown cells of the new facultative methylotrophs isolates donot convert secondary alcohols to methyl ketones.

Some enzymatic degradation of 2-butanone was observed. The product,2-butanone, did not inhibit the conversion of 2-butanol to thecorresponding 2-butanone. The rate of the 2-butanone production waslinear for the first four hours of incubation for the cultures tested.

A yeast culture had the highest production rate and had a highertemperature optimum (40° C.) and there was a reasonably high 2-butanoneproduction rate at 45° C. (The bacteria had a temperature optimum ofabout 35° C.)

Metal-chelating agents inhibit the production of 2-butanone whichsuggests the involvement of metal(s).

Secondary alcohol dehydrogenase activity was found in the cell-freesoluble extract of the sonically disrupted cells of the C₁ -grownisolates. The cell-free system requires a cofactor, specifically NAD,for its activity. The new secondary alcohol dehydrogenase specificallyand stoichiometrically oxidizes C₃ -C₆ secondary alcohols to theircorresponding methyl ketones. The enzyme has been purified 2,600 foldand shows a single protein band on acrylamide gel electrophoresis. Ithas a molecular weight of 95,000 dalton. The bacterial SADH consists oftwo subunits of 48,000 dalton and two atoms of zinc per molecule ofenzyme protein. Primary alcohls are not converted to ketones by theSADH. The pH and temperature optima for SADH are 8-9, and 30°-35° C.,respectively. The activation energy calculated is 19.8 K cal. Acrylamidegel electrophoresis of the purified SADH fraction stained with coomassiebrilliant blue and activity stain, as well as the crude solublecell-free extracts from distinct types of methanol-grown microbesstained with activity stain were compared. Both the protein stain andthe enzyme activity stain of the purified SADH showed a single proteinband. The mobility on the gel electrophoresis of SADH from the distincttypes of methanol-grown bacterial cells were identical. Yeast SADH hadfaster mobility toward anode on the gel electrophoresis. The addition ofsubstrates in the SADH reaction does not require an obligatory order.The SADH activity is inhibited by metal-chelating agents, by strongthio-reagents, and by the product 2-butanone.

Cell suspensions of yeasts of the invention grown on methyl radicaldonating compounds (e.g., methanol, methylamine, methyl formate, etc.)catalyze the conversion of secondary alcohols to the correspondingmethyl ketones.

Cell-free extracts derived from methyl-raadical (e.g., methanol)-grownyeasts of the invention catalyzed an NAD⁺ -dependent oxidation of C₃ -C₆secondary alcohols to the corresponding methyl ketones. The purifiedNAD⁺ -specific secondary alcohol dehydrogenase from methanol-grown yeastof the invention is homogeneous as judged by polyacrylamide gelelectrophoresis. The purified enzyme catalyzes the conversion ofsecondary alcohols to the corresponding methyl ketones in the presenceof NAD⁺ as an electron acceptor. Primary alcohols were not oxidized bythe purified enzyme. The optimum pH for conversion of secondary alcoholsby the purified yeast-derived enzyme is 8. The molecular weight of thepurified yeast-derived SADH as determined by gel filtration is 98,000and subunit size as determined by sodium dodecyl sulfate gelelectrophoresis is 48,000. The activity of the purified yeast-derivedSADH was inhibited by sulfhydryl inhibitors and metal-binding agents.

C₃ -C₆ n-alkanes are converted to C₃ -C₆ sec. alcohols by cellsuspensions of the methane-grown methylotrophs of of the invention andthe secondary alcohols accumulate extracellularly. Other microorganisms,e.g., yeasts, actinomycetes, and fungi, grown on C₁ -compounds willoxidize the C₃ -C₆ n-alkanes to the corresponding sec. alcohols.

C₃ -C₆ n-alkanes are converted to C₃ -C₆ sec. alcohols by cell-freeparticulate fractions derived from the methylotrophic microorganisms ofthe invention. The reaction requires oxygen and reduced nicotinamideadenine dinucleotide (NADH) as electron donor. The conversion of then-alkanes to the sec. alcohols is inhibited by thio-containing compoundsand metal-binding agents such as α,α-bipyridyl, thiosemicarbazide,thiourea, 1,10-phenanthroline, and 8-hydroxyquinoline. (This suggeststhe involvement of metal ion(s) in the oxidation of C₃ -C₆ n-alkanes tosec. alcohols.) The hydroxylation of C₃ -C₆ n-alkanes to thecorresponding sec. alcohols is inhibited in the presence of propylene.This suggests that the propylene and n-alkanes (e.g., propane) arecompeting for the same enzyme system(s). Ascorbate and reducednicotinamide adenine dinucleotide phosphate (NADPH) could also beutilized as electron donor in place of NADH for hydroxylation ofn-alkanes to the corresponding sec. alcohols.

The newly discovered and isolated methane and methyl-radical-utilizing(methylotrophic) microorganism strains of the present invention have thefollowing identifying characteristics:

                  TABLE I                                                         ______________________________________                                                                    U.S.D.A.                                          Methylotropic               Agriculture                                       Microorganism   ER & E      Research Center                                   Strain Name     Designation Designation                                       ______________________________________                                        1.   Methylosinus   (CRL 15 PM1)                                                                              NRRL B-11,202                                      trichosporium                                                            2.   Methylosinus   (CRL 16 PM2)                                                                              NRRL B-11,203                                      sporium                                                                  3.   Methylocystis  (CRL 18 PM4)                                                                              NRRL B-11,204                                      parvus                                                                   4.   Methylomonas   (CRL M4P)   NRRL B-11,205                                      methanica                                                                5.   Methylomonas   (CRL 21 PM7)                                                                              NRRL B-11,206                                      methanica                                                                6.   Methylomonas   (CRL M8Y)   NRRL B-11,207                                      albus                                                                    7.   Methylomonas   (CRL 17 PM3)                                                                              NRRL B-11,208                                      streptobacterium                                                         8.   Methylomonas   (CRL 22 PM9)                                                                              NRRL B-11,209                                      agile                                                                    9.   Methylomonas   (CRL M6P)   NRRL B-11,210                                      rubrum                                                                   10.  Methylomonas   (CRL 20 PM6)                                                                              NRRL B-11,211                                      rubrum                                                                   11.  Methylomonas   (CRL M10P)  NRRL B-11,212                                      rosaceus                                                                 12.  Methylomonas   (CRL M7P)   NRRL B-11,213                                      rosaceus                                                                 13.  Methylobacter  (CRL M6)    NRRL B-11,214                                      chroococcum                                                              14.  Methylobacter  (CRL 23 PM8)                                                                              NRRL B-11,215                                      chroococcum                                                              15.  Methylobacter  (CRL M1Y)   NRRL B-11,216                                      bovis                                                                    16.  Methylobacter  (CRL 19 PM5)                                                                              NRRL B-11,217                                      bovis                                                                    17.  Methylobacter  (CRL M5Y)   NRRL B-11,218                                      vinelandii                                                               18.  Methylococcus  (CRL M1)    NRRL B-11,219                                      capsulatus                                                               19.  Methylococcus  (CRL 24 PM12)                                                                             NRRL B-11,220                                      minimus                                                                  20.  Methylococcus  (CRL 25 PM13)                                                                             NRRL B-11,221                                      capsulatus                                                               21.  Methylobacterium                                                                             (CRL 26 R6) NRRL B-11,222                                      organophilum                                                             22.  Pichia sp.     (CRL-72)    NRRL Y-11,328                                 23.  Torulopsis sp. (A.sub.1)   NRRL Y-11,419                                 24.  Kloeckera sp.  (A.sub.2)   NRRL Y-11,420                                      and mutants therof.                                                      ______________________________________                                    

An important characteristic of the strains of the present invention (asidentified above) is their capability to produce microbial cells whencultured under aerobic conditions in a liquid growth medium comprisingassimilable sources of nitrogen and essential mineral salts in thepresence of methane gas or a methylradical donating carbon-containingcompounds such as methanol, methylamine, methyl formate, methylcarbonate, dimethyl ether, etc. as the major carbon and energy source.

The above strains have been deposited at the United States Department ofAgriculture, Agriculture Research Service, Northern Regional ResearchLaboratory (NRRL), Peoria, Ill. 61604 and have received from NRRL theindividual NRRL designations as indicated above pursuant to a contractbetween NRRL and the assigned of this patent application (Exxon Researchand Engineering Company (ER&E)). The contract with NRRL provides forpermanent availability of the progeny of these strains to the publicincluding citizens of West Germany, upon the issuance of the U.S. patentor the publication of a German patent application corresponding to thisapplication, whichever comes first occurs and that progemy of thesestrains will be made available to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accodingto 35 USC 122 and the Commissioner's rules pertaining thereto (including35 CRF 1.14, with particular reference to 886 OG 638) or the West GermanPatent Office. The assignee of the present application had agreed that,if any of these strains on deposit should die, or is destroyed, duringthe effective life of the patent, it will be replaced with a livingstrain of the same organism.

The Taxonomical characteristics of these newly isolated strains areshown below:

MORPHOLOGICAL AND TAXONOMICAL CHARACTERISTICS OF METHYLOTROPHICMICROORGANISMS

1. Methylosinus trichosporium strain CRL 15 PM1 (NRRL B-11,202) produceswhite round colonies on salt agar plates in the presence of CH₄ or CH₃OH. The organisms are motile, rod-shaped, gram-negative, aerobic.Rosettes frequently formed in old culture. Organisms form exospores.Grows at the expense of methane and methanol. Organic compounds otherthan C₁ compounds do not support growth. It has a Type I membranestructure.

2. Methylosinus sporium strain CRL 16 PM2 (NRRL B-11,203) Produces whitecolonies on salt agar plates in the presence of CH₄ or CH₃ OH. Theorganisms are motile, vibrio-shaped, gram-negative, aerobic. Rosettesfrequently formed. Organisms form exospores. Grows at the expense ofmethane and methanol. Organic compounds other than C₁ compounds do notsupport growth. It has a Type II membrane structure.

3. Methylocystis parvus strain CRL 18 PM4 (NRRL B-11,204) Producesmucoid white colonies on salt agar plates in the presence of CH₄ or CH₃OH. The organisms are non-motile, cocco-bacillus shaped, gram-negative,aerobic. Old cultures form cysts which are dessication-resistant. Growsat the expense of methane and methanol. Organic compounds other than C₁compounds do not support growth. It has a Type II membrane structure.

4. Methylomonas methanica CRL M4P (NRRL B-11,205) Produces pink raisedcolonies on salt agar plates in the presence of CH₄ or CH₃ OH. Theorganisms are motile, rod-shaped, gram-negative, aerobic. Produces slimycapsule. It has a Type I membrane structure.

5. Methylomonas methanica CRL 21 PM7 (NRRL B-11,206) Produces pinkcolonies on salt agar plates in the presence of CH₄ or CH₃ OH. Theorganisms are motile, rod-shaped, gram-negative, aerobic. Produces slimycapsule. Grows at the expense of methane and methanol. Organic compoundsother than C₁ compounds do not support growth. It has a Type I membranestructure.

6. Methylomonas albus CRL M8Y (NRRL B-11,207) Produces white to yellow(with age) and fuzzy-edged colonies on salt agar plates in the presenceof CH₄ or CH₃ OH. The organisms are rod-shaped, motile, gram-negative,aerobic. Produces slimy capsule. It has a Type I membrane structure.

7. Methylomonas streptobacterium CRL 17 PM3 (NRRL B-11,208) Produceswhite colonies on salt agar plates in the presence of CH₄ or CH₃ OH. Theorganisms are non-motile, rod-shaped, gram-negative, aerobic. Producesslimy capsule. Grows at the expense of methane and methanol. Organiccompounds other than C₁ compounds do not support growth. It has a Type Imembrane structure.

8. Methylomonas agile CRL 22 PM9 (NRRL B-11,209) Produces white colonieson salt agar plates in the presence of CH₄ or CH₃ OH. The organisms arenon-motile, rod-shaped, gram-negative, aerobic. Grows at the expense ofmethane and methanol. Organic compounds other than C₁ compounds do notsupport growth. It has a Type I membrane structure.

9. Methylomonas rubrum CRL M6P (NRRL B-11,210) Produces pink-oragne,circular and raised colonies on salt agar plates in the presence of CH₄or CH₃ OH. The organisms are rod-shaped, motile, gram-negative, aerobic.Produces slimy capsule. It has a Type I membrane structure.

10. Methylomonas rubrum CRL 20 PM6 (NRRL B-11,211) Produces red colonieson salt agar plates in the presence of CH₄ or CH₃ OH. The organisms aremotile, rod-shaped, gram-negative, aerobic. Produces slimy capsule.Grows at the expense of methane and methanol. Organic compounds otherthan C₁ compounds do not support growth. It has a Type I membranestructure.

11. Methylomonas rosaceus CRL M10P (NRRL B-11,212) Produces pink andcircular colonies on salt agar plates in the presence of Ch₄ or CH₃ OH.The organisms are motile, long thin rods, gram-negative, aerobic. It hasa Type I membrane structure.

12. Methylomonas rosaceus CRL M7P (NRRL B-11,213) Produces pink andfuzzy-edged colonies on salt agar plates in the presence of CH₄ or CH₃OH. The organisms are small thin rods, motile, gram-negative, aerobic.Produces slimy capsule. It has a Type I membrane structure.

13. Methylobacter chroococcum CRL M6 (NRRL B-11,214) Producescream-colored and fuzzy-edged colonies on salt agar plates in thepresence of CH₄ or CH₃ OH. The organisms are non-motile, largerlds/cocci, gram-negative, aerobic. Produces slimy capsule. It has aType I membrane structure.

14. Methylobacter chroococcum CRL 23 PM8 (NRRL B-11,215) Produces palepink colonies on salt agar plates in the presence of CH₄ or Ch₃ OH. Theorganisms are non-motile, gram-negative, aerobic. Produces slimycapsule. Grows at the expense of methane and methanol. Organic compoundsother than C₁ compounds do not support growth. It has a Type I membranestructure.

15. Methylobacter bovis CRL M1Y (NRRL B-11,216) Produces yellow,circular colonies on salt agar plates in the presence of CH₄ or CH₃ OH.The organisms are non-motile large rods, gram-negative, aerobic. It hasa Type I membrane structure.

16. Methylobacter bovis CRL 19 PM 5 (NRRL B-11,217) Produces white tobrown colonies on salt agar plates in the presence of CH₄ or CH₃ OH. Theorganisms are non-motile, gram-negative, aerobic. Produces slimycapsule. Grows at the expense of methane and methanol. Organic compoundsother than C₁ compounds do not support growth. It has a Type I membranestructure.

17. Methylobacter vinelandii CRL M5Y (NRRL B-11,218) Produces smalldistinct, light colored (to yellow with age)--colonies on salt agarplates in the presence of CH₄ or CH₃ OH. The organisms are motile,rod-shaped gram-negative, aerobic. It has a Type I membrane structure.

18. Methylococcus capsulatus CRL M1 (NRRL b-11,219) Produces whitecolonies on salt agar plates in the presence of CH₄ or CH₃ OH. Theorganisms are non-motile, cocci, gram-negative, aerobic. It has a Type Imembrane structure.

19. Methylococcus minimus CRL 24 PM12 (NRRL B-11,220) Produces whitecolonies on salt agar plates in the presence of CH₄ or CH₃ OH. Theorganisms are non-motile cocci, gram-negative, aerobic. Sometimesorganisms occur in chains and have the ability to grow at 37° and 45° C.Grows at the expense of methane and methanol. Organic compounds otherthan C₁ compounds do not support growth. It has a Type I membranestructure.

20. Methylococcus capsulatus CRL 25 PM13 (NRRL B-11,221) Produces whitecolonies on salt agar plates in the presence of CH₄ or CH₃ OH. Theorganisms are non-motile cocci, occurs in pairs, gram-negative, aerobic.Organisms form capsules and have the ability to grow at 37° and 45° C.Grows at the expense of methane and methanol. Organic compounds otherthan C₁ compounds do not support growth. It has a Type I membranestructure.

21. Methylobacterium organophilum CRL 26 R6 (NRRL B-11,222) Produceswhite colonies on salt agar plates in the presence of methane ormethanol. The organisms are motile, rod-shaped, gram-negative, aerobic.Grows at the expense of methane, methanol, glucose, succinate andnutrient agar. (Therefore, it is classically a facultative type). It hasa Type I membrane structure.

22. Pichia sp. CRL-72 (NRRL Y-11,328) Produces slimy white colonies onplates. Cells are large and oval; some cells have buds. Reproduce bybudding and they grow aerobically on C₁ -C₆ primary alcohols, C₁ -C₄primary amines, methyl formate, succinate and nutrient agar. They do notgrow on methane.

23. Torulopsis sp. A₁ (NRRL Y-11,419) Capable of growth on methanol,methyl formate, methylamine, ethanol, propylamine, and nutrient agar.Does not grow on methane. Cells are large oval shape and showmultilateral budding under microscopic examination.

24. Kloeckera sp. A₂ (NRRL Y-11,420) Capable of growth on methanol,methyl formate, methylamine, ethanol, propylamine, and nutrient agar.Does not grow on methane. Cells are large oval shape and show bipolarbudding under microscopic examination.

The newly discovered and isolated strains of the present invention wereobtained from soil samples from the Bayway Refinery in Linden, N.J., andfrom lake water samples from Warinaco Park, Linden, N.J., and fromRobert's Pond, Ridgefield, Conn. The samples were screened formethylotrophic microorganisms by growth under oxygen and methane. Themethylotrophs were then isolated, purified, and maintained by theprocedure described below.

The maintenance of the cultures of these newly discovered and isolatedstrains should be carefully controlled. The preferred means formaintaining the cultures is described below in Table II.

                  TABLE II                                                        ______________________________________                                        MAINTENANCE OF CULTURES                                                       The organisms are perferably subcultured every                                two weeks on mineral salts agar plates which contain                          medium having the following composition:                                      ______________________________________                                        Na.sub.2 HPO.sub.4    0.21 g                                                  NaH.sub.2 PO.sub.4    0.09 g                                                  NaNO.sub.3            2.0 g                                                   MgSO.sub.4 . 7H.sub.2 O                                                                             0.2 g                                                   KCl                   0.04 g                                                  CaCl.sub.2            0.015 g                                                 FeSO.sub.4 . 7H.sub.2 O                                                                             1 mg                                                    CuSO.sub.4 . 5H.sub.2 O                                                                             0.01 mg                                                 H.sub.3 BO.sub.4      0.02 mg                                                 MnSO.sub.4 . 5H.sub.2 O                                                                             0.14 mg                                                 ZnSO.sub.4            0.02 mg                                                 MoO.sub.3             0.02 mg                                                 Agar                  15 g                                                    Water                 1 liter                                                 ______________________________________                                    

In the case of yeast cells, yeast nitrogen base is added to the abovemedium.

These plates should be incubated in glass dessicators which have lidswith an airtight seal and external sleeves with a tooled hoseconnection. Dessicators are to be evacuated and filled with a gasmixture of methane and air (1:1 v/v). Incubation should be at 30° C.Cultures will survive in these dessicators for three months at 4° C.However, frequent transfer of cultures is preferred.

In commercial processes for the propagation of microorganisms, it isgenerally necessary to proceed by stages. These stages may be few ormany, depending on the nature of the process and the characteristics ofthe microorganisms. Ordinarily, propagation is started by inoculatingcells from a slant of a culture into a pre-sterilized nutrient mediumusually contained in a flask. In the flask, growth of the microorganismsis encouraged by various means, e.g., shaking for thorough aeration, andmaintenance of suitable temperature. This step or stage is repeated oneor more times in flasks or vessels containing the same or larger volumesof nutrient medium. These stages may be conveniently referred to asculture development stages. The microorganisms with or withoutaccompanying culture medium, from the last development stage, areintroduced or inoculated into a large scale fermentor to producecommercial quantities of the microorganisms or enzymes therefrom.

Reasons for growing the microorganisms in stages are manyfold, but areprimary dependent upon the conditions necessary for the growth of themicroorganisms and/or the production of enzymes therefrom. These includestability of the microorganisms, proper nutrients, pH, osmoticrelationships, degree of aeration, temperature and the maintenance ofpure culture conditions during fermentation. For instance, to obtainmaximum yields of the microbial cells, the conditions of fermentation inthe final stage may have to be changed somewhat from those practiced toobtain growth of the microorganisms in the culture development stages.Maintaining the purity of the medium, also, is an extremely importantconsideration, especially where the fermentation is performed underaerobic conditions as in the case of the methylotroph microorganisms. Ifthe fermentation is initially started in a large fermentor, a relativelylong period of time will be needed to achieve an appreciable yield ofmicroorganisms and/or oxidative and dehydrogenase enzymes therefrom.This, of course, enhances the possibility of contamination of the mediumand mutation of the microorganisms.

The culture media used for growing the methylotrophic microorganisms andinducing the oxidative enzyme system will be comprised of inorganicsalts of phosphate, sulfates and nitrates as well as oxygen and a sourceof C₁ compounds. The fermentation will generally be conducted attemperatures ranging from 5° to about 50° C., preferably at temperaturesranging from about 25° C. to about 45° C. The pH of the culture mediumshould be controlled at a pH ranging from about 4 to 9 and preferablyfrom about 5.5 to 8.5 and more preferably from 6.0 to 7.5. Thefermentation may be conducted at atmospheric pressures although higherpressures up to about 5 atmospheres and higher may be employed.

Typically, to grow the methylotrophic microorganisms and to induce theoxygenase and dehydrogenate enzymes, the microorganisms are inoculatedinto the medium which is contacted with a gas mixture containing methaneand oxygen. Methane may be supplied in the form of natural gas. Forcontinuous flow culture the microorganisms may be grown in any suitablyadapted fermentation vessel, for example, a stirred baffled fermentor orsparged tower fermentor, which is provided either with internal coolingor an external recycle cooling loop. Fresh medium may be continuouslypumped into the culture at rated equivalent to 0.02 to 1 culture volumeper hour and the culture may be removed at a rate such that the volumeof culture remains constant. A gas mixture containing methane and oxygenand possibly carbon dioxide or other gases is contacted with the mediumpreferably by bubbling continuously through a sparger at the base of thevessel. The source of oxygen for the culture may be air, oxygen oroxygen-enriched air. Spent gas may be removed from the head of thevessel. The spent gas may be recycled either through an external loop orinternally by means of a gas inducer impeller. The gas flows and recycleshould be arranged to give maximum growth of microorganism and maximumutilization of methane.

The oxygenase enzyme system may be obtained, as described above, as acrude extract, or a cell-free particulate fraction, i.e., the materialwhich precipitates when the supernatant after centrifuging broken cellsat 10,000×g. for 30 min. is centrifuged for 1 hour at 10,000×g. orgreater. When it is desired to obtain the secondary alcoholdehydrogenase (SADH) enzyme fraction one first breaks the cells, e.g.,sonication, etc., secondly removes the cellular debris, e.g.,centrifuges at 10,000×g. for about 20 minutes and the recovered crudeSADH enzyme can thereafter be further purified by mild heat treatment,column chromatography, etc., as described in the examples below. Themicrobial cells may be harvested from the growth medium by any of thestandard techniques commonly used, for example, flocculation,sedimentation, and/or precipitation, followed by centrifugation and/orfiltration. The biomass may be dried, e.g., by freeze or spray dryingand may be used in this form for further use in the oxidation reactions.

To put the invention to practice, an oxaditive or dehydrogenase enzymepreparation is obtained, such as, for example, in the manner describedabove, which will convert methane to methanol under oxidativeconditions. It is preferred to obtain such a preparation from one of themicroorganism strains (or natural and/or artificial mutant thereof)described above and grow the microorganism in a nutrient mediumcontaining methane and oxygen as described above. The nutrient mediummay be the one described by Whittenbury et al. or more preferably theculture medium described by Foster and Davis, J. Bacteriol., 91,1924-1931 (1966). In the case of yeast, a yeast nitrogen base is added.

The enzyme preparation is then brought into contact with the desiredoxidizable substrate, e.g., a C₂ -C₄ alkene, e.g., ethylene, propylene,butene-1 or conjugated butadiene or mixtures thereof, a cyclic compoundsuch as cyclohexane, an alkane such as methane, ethane, propane orbutane, etc., or a secondary alcohol, e.g., 2-propanol or 2-butanol inthe presence of oxygen and a buffer solution or nutrient medium (e.g.,the same nutrient medium used to produce the microorganism may be usedexcept that the oxidizable substrate material has replaced the methane)and the mixture is incubated until the desired degree of conversion hasbeen obtained. Thereafter, the oxidized roduct is recovered byconventional means, e.g., distillation, etc.

To facilitate the necessary effective contact of oxygen and the enzyme(whether it be an enzyme preparation or methylotrophic microorganisms),it is preferred, for best results, to employ a strong, finely dividedair stream into a vigorously stirred dispersion of substrate in theoxidation medium that generally contains water, and a buffer which theenzyme preparation or microorganism culture is suspended. The enzymepreparation may then be separated from the liquid medium, preferably byfiltration or centrifugation. The resulting oxidized product may thengenerally be obtained.

The process of the invention may be carried out batchwise,semi-continuously, continuously, concurrently or countercurrently.Optionally, the suspension containing the enzyme preparation ormethylotrohic microorganisms and buffer solution is passed downwardlywith vigorous stirring countercurrently to an air stream rising in atube reactor. The top layer is removed from the downflowing suspension,while culture and remaining buffer solution constituents are recycled,at least partly, with more oxidative substrate and addition of freshenzyme preparation or methylotrophic microorganisms, as required.

The growth of the methylotrophic microorganisms and the oxidationprocess may be conveniently coupled by conducting them simultaneously,but separately and using much higher aeration in the oxidation process(e.g., an air excess of at least twice that required for growth,preferably at least five times as much aeration). Both the growthprocess and the methane hydroxylation or oxidation processes may beconducted in the same reactor in sequential or simultaneous operationsby alternate use of normal and strong aeration.

The invention is illustrated further by the following examples which,however, are not to be taken as limiting in any respect. All parts andpercentages, unless expressly stated otherwise, are by weight.

EXAMPLE 1

A nutrient medium as described by Foster and Davis, J. Bacteriol., 91,1924-1931 (1966) having the following composition per liter wasprepared:

    ______________________________________                                        Na.sub.2 HPO.sub.4    0.21 g.                                                 NaH.sub.2 PO.sub.4    0.09 g.                                                 NaNO.sub.3            2.0 g.                                                  MgSO.sub.4 . 7H.sub.2 O                                                                             0.2 g.                                                  KCl                   0.04 g.                                                 CaCl.sub.2            0.015 g.                                                FeSO.sub.4 . 7H.sub.2 O                                                                             1 mg.                                                   CuSO.sub.4 . 5H.sub.2 O                                                                             0.01 mg.                                                H.sub.3 BO.sub.4      0.02 mg.                                                MnSO.sub.4 . 5H.sub.2 O                                                                             0.02 mg.                                                ZnSO.sub.4            0.14 mg.                                                MoO.sub.3             0.02 mg.                                                ______________________________________                                    

The pH of the nutrient medium was adjusted to 7.0 by the addition ofacid or base and 50 ml samples of the nutrient medium was charged into aplurality of 300 ml shaker flasks. The shaker flasks were inoculatedwith an inoculating loop of cells from an agar plate containinghomogeneous colonies of the microorganisms on the plate (the purity ofthe isolates was confirmed by microscopic examination). The isolates hadbeen maintained on agar plates under an atmosphere of methane and airhaving a 1:1 v/v gas ratio which had been transferred every two weeks.The gaseous phase of the inoculated flasks was then replaced with a gasmixture comprised of methane and air having a ratio of 1:1 on a v/vbasis. The inoculated flasks were sealed air tight and were incubated ona rotary shaker of orbital radius 2.5 cm at 250 rpm and at 30° C. fortwo days until turbidity in the medium had developed.

The cells were harvested by centrifugation at 10,000×g. at 4° C. for 30minutes. The cell pellet was washed twice with a 0.15 M phosphate bufferat a pH of 7.0 (containing 0.002 M MgCl₂). The washed cells were thensuspended in a 0.15 M phosphate buffer at pH 7.0.

A 0.5 ml sample of each washed cell suspension (2 mg cells) was put into10 ml vials at 4° C. which were sealed with a rubber cap. The gaseousphase of the vials was removed with vacuum and then was replaced with agas mixture of the oxidative substrate (e.g., methane) and oxygen at a1:1 v/v ratio. The vials were then incubated at 30° C. on a rotaryshaker at 300 rpm. Samples of product (3 μl) were withdrawn periodicallywith a microsyringe and the products were analyzed by gas chromatography(ionization flame detector column).

The newly discovered and isolated microorganism strains of the presentinvention were each grown aerobically in the presence of methane in themanner described above. The methane-grown microbial cells were thenwashed, recovered and put to use in oxidizing an oxidative substrate bythe procedure described above except in the case of a liquid substrate10 μl of the substrate were used. Table III shows the results of theseexperiments where the oxidative substrates were methane, ethylene,propylene, butene-1 and butadiene. The methane-grown methylotrophicmicroorganisms did not produce any detectable epoxide product frompentene-1 or hexene-1.

                                      TABLE III                                   __________________________________________________________________________    Oxidation Conversion Rates For Methane and Lower                              Alkenes With Methylotrophic Microorganisms                                                   Oxidative Conversion Rates (μmole/hr/mg                                    protein).sup.(b)                                                                   Ethylene                                                                           Propylene                                                                           Butene-1                                                                            Butadiene                                               Methane                                                                            to   to    to    to                                       Methylotrophic Microorganism                                                                 to   Ethylene                                                                           Propylene                                                                           1,2-epoxy-                                                                          1,2-epoxy-                               Strain Identification.sup.(a)                                                                Methanol                                                                           Oxide                                                                              Oxide butane                                                                              butene                                   __________________________________________________________________________    Methylosinus trichosporium                                                                   1.2  2.5  4.5   1.8   4.2                                      (CRL 15 PM1) NRRL B-11,202                                                    Methylosinus sporium                                                                         1.0  1.3  3.2   1.2   2.5                                      (CRL 16 PM2) NRRL B-11,203                                                    Methylocystis parvus                                                                         --   0.9  1.5   --    --                                       (CRL 18 PM4) NRRL B-11,204                                                    Methylomonas methanica                                                                       2.3  1.0  3.8   2.8   1.0                                      (CRL M4P) NRRL B-11,205                                                       Methylomonas methanica                                                                       --   2.1  2.4   --    --                                       (CRL 21 PM7) NRRL B-11,206                                                    Methylomonas albus                                                                           1.5  1.8  2.5   1.8   3.0                                      (CRL M8Y) NRRL B-11,207                                                       Methylomonas streptobacterium                                                                1.1  0.7  1.0   2.5   2.6                                      (CRL 17 PM3) NRRL B-11,208                                                    Methylomonas agile                                                                           0.6  2.2  1.6   0.5   2.0                                      (CRL 22 PM9) NRRL B-11,209                                                    Methylomonas rubrum                                                                          0.5  0.7  1.0   --    --                                       (CRL M6P) NRRL B-11,210                                                       Methylomonas rubrum                                                                           0.42                                                                              1.1  1.9   --    --                                       (CRL 20 PM6) NRRL B-11,211                                                    Methylomonas rosaceus                                                                        7.3  4.7  1.41  3.1   9.2                                      (CRL M10P) NRRL B-11,212                                                      Methylmonas rosaceus                                                                         1.4  2.9  2.0   --    --                                       (CRL M7P) NRRL B-11,213                                                       Methylobacter chroococcum                                                                    0.8  1.6  1.2   0.7   1.5                                      (CRL M6) NRRL B-11,214                                                        Methylobacter chroococcum                                                                     0.72                                                                              1.2  2.5    0.82 1.8                                      (CRL 23 PM8) NRRL B-11,215                                                    Methylobacter bovis                                                                          1.5  0.64 1.5   1.1    1.27                                    (CRL M1Y) NRRL B-11,216                                                       Methylobacter bovis                                                                          2.0  1.3  2.2   1.3   2.3                                      (CRL 19 PM5) NRRL B-11,217                                                    Methylobacter vinelandii                                                                     0.9  1.2  1.9   0     --                                       (CRL M5Y) NRRL B-11,218                                                       Methylococcus capsulatus                                                                     2.5  5.7  5.5   1.3   4.4                                      (CRL M1) NRRL B-11,219                                                        Methylococcus minimus                                                                        2.3  1.2  1.8   1.5   3.0                                      (CRL 24 PM12) NRRL B-11,220                                                   Methylococcus capsulatus                                                                     2.1  0.8  1.2   1.9   2.5                                      (CRL 25 PM13) NRRL B-11,221                                                   Methylobacterium organophilum                                                                0.7  0.9  2.5   0.9   2.8                                      (CRL 26 R6) NRRL B-11,222                                                     __________________________________________________________________________     .sup.(a) The dry weight of the cells was about 0.2 g/100 ml culture broth     .sup.(b) The products of microbial oxidation were identified by gas           chromatographic retention time with authenic standards. The identificatio     of the products was supplemented by establishing the presence or absence      of product peaks before and after bromination or acid hydrolysis. Analysi     also revealed that no further oxidation of epoxide product occurred.     

In comparative experimental tests, washed cell suspensions of themethylotrophic microorganism strains of the present invention grown onmethanol did not possess the ability to either hydroxylate methane orthe ability to epoxidize the C₂ -C₄ alkenes.

EXAMPLE 2

The procedure of Example 1 was repeated using Methylobacter bovis CRLMlY NRRL B-11,216 and Methylococcus capsulatus CRL Ml NRRL B-11,219except that the microorganisms were grown in the aerobic methanenutrient medium at 40° C. The washed cells of the methane-grown cellswere then contacted with propylene by the procedure described in Example1 (30° C. reaction temperature) and the reaction product (propyleneoxide) was analyzed by gas chromatography retention time. Themethane-grown methylotrophic microorganisms (Methylobacter bovis CRL MlYNRRL B-11,216 and Methylococcus capsulatus CRL Ml NRRL B-11,219converted propylene to propylene oxide at a conversion rate of 0.27μmole/2hr./mg. protein and 7.88 μmole/2 hr./mg. protein, respectively,wherein the dry weight of the cells was about 0.2 g./100 ml. of culturebroth.

EXAMPLE 3 Microbiological Conversion of Sec. Alcohols to Ketones

The procedure in Example 1 was repeated wherein a plurality of the newlydiscovered and isolated strains were each grown aerobically in themethane-containing nutrient medium. The washed cells of themethane-grown methylotrophic microorganisms were then contacted withsec. alcohols, i.e., isopropanol or 2-butanol by the oxidation procedureof Example 1. The reaction products were analyzed and found to containacetone and 2-butanone, respectively. The results of this series ofexperiments are shown in Table IV.

                  TABLE IV                                                        ______________________________________                                        Microbiological Conversion of Sec. Alcohols to Ketones                                           Conversion Rate                                                               to Ketone   Product                                                           (μmole/2 Formed                                         Methylotrophic Microorganism                                                                     mg protein) (detected                                      Strain Identification                                                                            2 hrs.  20 hrs. by GC)                                     ______________________________________                                            Methane-Grown-                                                                Isopropanol Substrate                                                     1.  Methylobactor chroococcum                                                                        1       8     Acetone                                      (CRL M6) NRRL B-11,214                                                    2.  Methylococcus capsulatus                                                                         3       15    Acetone                                      (CRL M1) NRRL B-11,219                                                    3.  Methylosinus trichosporium                                                                       3       --    Acetone                                      (CRL 15 PM1) NRRL                                                             B-11,202                                                                  4.  Methylomonas rubrum                                                                              1       --    Acetone                                      (CRL M6P) NRRL B-11,210                                                   5.  Methylobacter bovis                                                                              5       --    Acetone                                      (CRL M1Y) NRRL B-11,216                                                   6.  Methylobacter organophilum                                                                       3       --    Acetone                                      (CRL 26 R6) NRRL B-11,222                                                     Methane-Grown-                                                                2-Butanol Substrate                                                       7.  Methylosinus trichosporium                                                                       20      --    2-Butanone                                   (CRL 15 PM1) NRRL                                                             B-11,202                                                                  8.  Methylomonas methanica                                                                           0.06    2     2-Butanone                                   (CRL M4P) NRRL                                                                B-11,205                                                                  9.  Methylomonas rosaceus                                                                            --      --    --                                           (CRL M7P) NRRL B-11,213                                                   10. Methylobacter chroococcum                                                                        1       3     2-Butanone                                   (CRL M6) NRRL B-11,214                                                    11. Methylobacter bovis                                                                              20            2-Butanone                                   (CRL M1Y) NRRL                                                                B-11,216                                                                  12. Methylobacter vinelandii                                                                         2       18    2-Butanone                                   (CRL M5Y) NRRL B-11,218                                                   13. Methylococus capsulatus                                                                          18      50    2-Butanone                                   (CRL M1) NRRL B-11,219                                                    14. Methylobacterium organophilum                                                                    10      --    2-Butanone                                   (CRL 26 R6) NRRL B-11,222                                                 ______________________________________                                    

EXAMPLE 4 Microbiological Conversion of Alkanes to Ketones

A few of the newly discovered and isolated methylotrophic microorganismsof the invention were grown aerobically in a nutrient medium containingmethane in the manner described in Example 1 and the washed cells wereused to convert propane to acetone and butane to 2-butanone using thesame procedure for the substrate oxidation as described in Example 1.The results of these experiments are shown in Table V.

                  TABLE V                                                         ______________________________________                                        Microbiological Conversion of Alkanes to Ketones                                                Conversion                                                                    Rate                                                                          to Ketone                                                                     (μmoles/                                                 Methylotrophic Microorganism                                                                    hr/mg              Pro-                                     Strain Identification                                                                           Protein   Substrate                                                                              duct                                     ______________________________________                                        Methylobacter chroococcum                                                                       2.0       Propane  Ace-                                     (CRL M6) NRRL B-11,214               tone                                     Methylobacter bovis                                                                             5.0       Propane  Ace-                                     (CRL M1Y) NRRL B-11,216              tone                                     Methylococcus capsulatus                                                                        4.0       Propane  Ace-                                     (CRL M1) NRRL B-11,219               tone                                     Methylobacterium organophilum                                                                   3.0       Propane  Ace-                                     (CRL R6) NRRL B-11,222               tone                                     Methylosinus trichosporium                                                                      1.6       Propane  Ace-                                     (CRL 15 PM1) NRRL B-11,202           tone                                     Methylomonas rubrum                                                                             3.5       Propane  Ace-                                     (CRL 20 PM6) NRRL B-11,211           tone                                     Methylobacter chroococcum                                                                       1         Butane   2-Buta-                                  (CRL M6) NRRL B-11,214               none                                     Methylobacter bovis                                                                             3         Butane   2-Buta-                                  (CRL M1Y) NRRL B-11,216              none                                     Methylococcus capsulatus                                                                        2         Butane   2-Buta-                                  (CRL M1) NRRL B-11,219               none                                     Methylobacterium organophilum                                                                   2.0       Butane   2-Buta-                                  (CRL R6) NRRL B-11,222               none                                     Methylosinus trichosporium                                                                      1.0       Butane   2-Buta-                                  (CRL 15 PM1) NRRL B-11,202           none                                     Methylomonas methanica                                                                          1.5       Butane   2-Buta-                                  (CRL 21 PM7) NRRL B-11,206           none                                     Methylocystis parvus                                                                            1.0       Butane   2-Buta-                                  (CRL 18 PM4) NRRL B-11,203           none                                     ______________________________________                                    

EXAMPLE 5 Microbiological Conversion of n-Pentane and n-Hexane to MethylKetones by Cell-Suspensions of Methane-Grown MethylotrophicMicroorganisms

In this example, the procedure of Example 1 was repeated wherein aplurality of the methane-utilizing methylotrophic microorganisms of theinvention were each aerobically grown in a methane-containing nutrientmedium. The nutrients in the medium were the same as indicated inExample 1 wherein methane was used as the oxygenase enzyme inducer andmajor source of carbon and energy for growth. Following growth, thecells were harvested and washed as described in Example 1. The restingcells of the induced methane-grown methylotrophic microorganisms werethen contacted with n-pentane or n-hexane under aerobic conditions in abuffered solution by the procedure in Example 1. The results of thisseries of experiments are shown in Table VI.

                  TABLE VI                                                        ______________________________________                                        Microbiological Conversion of n-Pentane and n-Hexane to                       Methyl Ketones By Cell-Suspensions of Methane-Grown                           Methane-Utilizing Methylotrophic Microorganisms                                               Conversion Rate                                                               μmole/hr/mg protein.sup.(a)                                Methylotrophic Microorganism                                                                    n-Pentane to                                                                              n-Hexane to                                     Strain Identification.sup.(b)                                                                   2-Pentanone 2-Hexanone                                      ______________________________________                                        Methylosinus trichosporium                                                                      0.08        0.04                                            CRL 15 NRRL B-11,202                                                          Methylomonas methanica                                                                          0.06        0.01                                            CRL 21 NRRL B-11,206                                                          Methylobacter vinelandii                                                                        0.04        0.01                                            CRL M5Y NRRL B-11,218                                                         Methylococcus capsulatus                                                                        0.15        0.08                                            CRL M1 NRRL B-11,219                                                          Methylobacterium organophilum                                                                   0.18        0.02                                            CRL 26 NRRL B-11,222                                                          ______________________________________                                         .sup.(a) The products were identified by gas chromatography retention tim     comparisons with authentic standards. Analysis revealed that no further       oxidation of the products occurred.                                           .sup.(b) The dry weight of the cells was about 0.2 g/100 ml of culture        broth.                                                                   

EXAMPLE 6 Microbiological Conversion of C₃ -C₆ Linear Secondary Alcoholsto Methyl Ketones by Cell-Suspensions of Methanol-GrownMethane-Utilizing Methylotrophic Microorganisms

In this example, the procedure of Example 1 was repeated except that aplurality of the methane-utilizing methylotrophic microorganisms of theinvention were each aerobically grown in a methanol-containing nutrientmedium instead of a methane-containing medium. The nutrients in themedium were the same as indicated in Example 1 except that 0.4% v/vmethanol was used as the alcohol dehydrogenase enzyme inducer and majorsource of carbon and energy for growth. Following growth the cells wereharvested and washed as described in Example 1. The resting cells of theinduced methanol-grown methylotrophic microorganisms were then contactedwith a secondary alcohol under aerobic conditions in a buffered solutionby the procedure described in Example 1. The results of this series ofexperiments are shown in Table VII.

                  TABLE VII                                                       ______________________________________                                        Microbiological Conversion of C.sub.3 -C.sub.6 Secondary Alcohols to          Methyl Ketones By Cell-Suspensions of Methanol-Grown Methane-                 Utilizing Methylotrophic Microorganisms                                                      Conversion Rates.sup.(a)                                                      μmoles/hr/mg protein                                                                         2-Pent-                                                       2-Pro-  2-Buta- anol  2-Hex-                                                  panol   nol to  to 2- anol to                                Methylotrophic Microorganism                                                                   to Ace- 2-Buta- Penta-                                                                              2-Hex-                                 Strain Identification.sup.(b)                                                                  tone    none    none  anone                                  ______________________________________                                        Methylosinus trichosporium                                                                     0.50    4.5     0.09  0.06                                   CRL 15 NRRL B-11,202                                                          Methylocystis parvus                                                                           0.25    1.0     0.07  0.05                                   CRL 18 NRRL B-11,204                                                          Methylomonas methanica                                                                         2.0     2.5     0.05  0.03                                   CRL M4P NRRL B-11,205                                                         Methylomonas streptobacterium                                                                  0.67    2.0     --    --                                     CRL 17 PM3 NRRL B-11,208                                                      Methylobacter chroococcum                                                                      1.0     1.4     0.08  0.02                                   CRL M6 NRRL B-11,214                                                          Methylobacter bovis                                                                            0.40    1.8     --    --                                     CRL 19 NRRL B-11,217                                                          Methylobacter vinelandii                                                                       2.0     2.0     0.05  0.02                                   CRL M5Y NRRL B-11,218                                                         Methylococcus capsulatus                                                                       5.0     2.0     0.24  0.08                                   CRL M1 NRRL B-11,219                                                          Methylococcus capsulatus                                                                       0.62    0.95    --    --                                     CRL 25 NRRL B-11,221                                                          Methylobacterium organophilum                                                                  0.72    2.5     1.0   0.09                                   CRL 26 NRRL B-11,222                                                          ______________________________________                                         .sup.(a) The products were identified by gas chromatography retention tim     comparisons with authentic standards. Analysis also revealed that no          further oxidation of the products occurred.                                   .sup.(b) The dry weight of the cells was about 0.2 g/100 ml culture broth                                                                              

EXAMPLE 7 Microbiological Oxidation of Cyclic Hydrocarbons

Methylobacter vinelandii (CRL M5Y NRRL B-11,218) was aerobically grownin a methane-containing nutrient medium in the manner described inExample 1 and the methane-grown washed cells were contacted underaerobic conditions with cyclohexane, benzene and toluene. Cyclohexanewas found to be converted to cyclohexanol at a conversion rate of 1.5μmoles/2 hr./mg. protein, whereas no oxidative conversion was detectedin the case of benzene and toluene. Several of the other strains of thepresent invention which had been grown on methane did not produce anydetectable cyclohexanol when the methane-grown cells were contacted withcyclohexane.

The newly discovered and isolated microorganisms of the presentinvention grow well in a nutrient medium under aerobic conditionscontaining carbon-containing compounds. When the carbon-containingcompounds are oxygenase and/or alcohol dehydrogenase enzyme inducers,the resulting resting microbial cells and/or their enzyme preparationsare capable of increasing the oxidative state of a plurality ofoxidizable compounds (substrates). When methane is used as the oxygenaseand/or alcohol dehydrogenase enzyme inducer and the major carbon andenergy source, the resulting resting microbial cells and/or their enzymepreparations are capable of converting: C₁ -C₆ n-alkanes to alcohols andketones; C₂ -C₄ alkenes to the corresponding 1,2-epoxides; C₃ -C₆ linearsecondary alcohols to the corresponding methyl ketones; and microbialcells from at least one strain was capable of converting cyclohexane tocyclohexanol.

This capability of carrying out these useful oxidative and/or alcoholdehydrogenating conversions has been shown above with respect to boththe obligate and facultative methylotrophic microorganisms of theinvention. In the case of either the obligate or facultativemethylotrophic microorganisms of the present invention which have beenaerobically grown in a nutrient medium containing a methyl-radicaldonating or precursor compound such as methanol, methylamine or methylformate as the alcohol dehydrogenase enzyme inducer, the resultingresting microbial cells or their enzyme preparations are only capable ofconverting C₃ -C₆ linear secondary alcohols to the corresponding methylketones. These induced enzymes are not capable of converting the C₃ -C₆alkanes to the corresponding methyl ketones and they are not capable ofconverting the C₂ -C₄ alkenes to the corresponding 1,2-epoxides. Inbatch experiments of the epoxidation process using resting,methane-grown cells, the epoxidation reaction proceeds linearly for atleast two hours. In the case of ketone production the reaction proceededlinearly for at least 4 hours. The oxidative enzyme system(s) of themethane- or methand-grown microorganisms of the present invention isinducible (by the methane or methanol) and the oxidation productsaccumulate extracellularly (i.e., after the reaction, the reactionmixtures were centrifuged and the desired oxidation product was onlyfound in the supernatant fraction and not in the cell pellet).

As will be shown by the examples that follow, the methane-grownmicrobial cells and their enzyme preparations (including cell-freeextracts) possess both oxygenase and alcohol dehydrogenase enzymeactivity. It is believed that the methane itself induces the oxygenaseenzyme activity and the methanol resulting from the oxidation of methaneby the methylotrophic microorganism during growth induces the alcoholdehydrogenase enzyme. The induced oxygenase enzyme is responsible forconverting the C₃ -C₆ alkane to an intermediate oxidation product, andsecondary alcohol, whereas the induced alcohol dehydrogenase enzymedehydrogenates the secondary alcohol to the corresponding methyl ketone.Similarly, the monookygenase enzyme system induced by the methane growthsubstrate is at least partly responsible for catalyzing the conversionof the C₂ -C₄ alkenes and butadiene to the corresponding 1,2-epoxides.

The Epoxidation System--Cell-Free Extracts

As previously indicated both the whole cells and the cell-free extractscontaining the oxygenase enzyme activity of the methane grownmethylotrophs may be used in the hydroxylation and epoxidation reactionsin the presence of air. NADH and metal (iron or copper) may be added toenhance activity when the cell-free or pure enzyme preparations areused. In utilizing the cell-free enzyme system of the invention theenzyme preparations were prepared as follows.

Preparation of Cellular Fractions

Organisms were grown at 30° C. in 2.8 liter flasks containing 700 mlmineral salts medium as described in Example 1 with methane (methane andair, 1:1 parts by volume) as the sole carbon and energy source. Cellswere harvested during exponential growth by centrifugation at 12,000×g.for 15 min. at 4° C. Cells were washed twice with 25 mM potassiumphosphate buffer, pH 7.0 containing 5 mM MgCl₂. Cells were suspended inthe same buffer. The cell suspensions at 4° C. were disintegrated by asingle passage through a French Pressure cell (15,000 lb./in.²) andcentrifuged at 5000×g. for 15 min. to remove unbroken bacteria. Thesupernatant solution (crude extract) was then centrifuged at 40,000×g.for 30 min., yielding particulate P(40) and soluble S(40) fractions. TheS(40) fraction was subsequently centrifuged at 80,000×g. for 60 min.,yielding particulate P(80) and soluble S(80) fractions. The particulatefractions [P(40) and P(80)] were suspended in 25 mM potassium phosphatebuffer, pH 7.0, containing 5 mM MgCl₂ and homogenized at 4° C.

Enzyme Assay

The oxidation of methane and propylene by particulate [(P)40 and(P)80]fractions and soluble [S(80)] fraction was measured at 30° C. byestimating the production of methanol and propylene oxide, respectively.The reaction mixtures contained in 1.0 ml: 150 mM potassium phosphatebuffer, pH 7.0 containing 5 mM MgCl₂, 0.6 ml; 10 μmoles NADH, andcellular fraction.

Reaction mixtures were contained in 10 ml vials at 4° C. Vials weresealed with rubber caps. The gaseous phase in the vials was removedusing vacuum and then was replaced with a gas mixture of methane orpropylene and oxygen at a 1:1, v/v ratio. Oxidation of other gaseousn-alkanes and n-alkenes was examined as described above. For liquidsubstrates, 10 μl of substrate was used directly. Vials were thenincubated at 30° C. on a rotary shaker at 200 RPM.

The products of epoxidation of n-alkenes and hydroxylation of n-alkaneswere assayed by flame ionization gas chromatography using a stainlesssteel column (12'×1/8") packed with 10% Carbowax 20 M on 80/100Chromosorb W and Porapak Q column. The column temperature was maintainedisothermally at 120° C. The carrier gas flow rate was 30 ml/min. ofhelium. The various products were identified by retention timecomparisons and co-chromatography with authenic standards.

Specific activities were expressed as μmoles of products formed per hourper mg. protein. Concentrations of protein in various fractions weredetermined by the method of Lowry et al., J. Biol. Chem., 193: 265-275(1951).

Distribution of n-Alkanes- and n-Alkenes-Oxidizing Activities inCell-Fractions

Three distinct groups of methane-utilizing organisms were selected toexamine oxidation of n-alkanes (C₁ -C₄) and n-alkenes (C₂ -C₄) incell-free systems. Cellular fractions were prepared from Type I obligatemethane-utilizing organisms, Methylomonas sp. (CRL-17, NRRL B-11,208)and Methylococcus capsulatus (Texas, ATCC 19,069); Type II obligatemethane-utilizing organisms, Methylosinus trichosporium (OB3b, NRRLB-11,196) and Methylosinus sp. (CRL-15, NRRL B-11,202); and afacultative methane-utilizing bacterium, Methylobacterium sp. (CRL-26,NRRL B-11,222).

Table VIII shows the distribution of the methane-and propylene-oxidizingactivity in various fractions derived from these organisms. About 85-90%of the total activity was detected in the P(40) fraction and 10% wasdetected in the P(80) fraction. The soluble fraction S(80) did notcontain any activity. The specific activities for the methane and thepropylene oxidation in fractions P(40) and P(80) did not varysignificantly in the various organisms examined (Table IX). Epoxidationof propylene and hydroxylation of methane were both dependent upon thepresence of oxygen and NADH. NADPH or ascorbate and other electroncarriers could also be utilized. Both reactions were linear during thefirst 15 min. as measured by detection of product by gas chromatography.

                  TABLE VIII                                                      ______________________________________                                        DISTRIBUTION OF PROPYLENE - AND METHANE-                                      OXIDIZING ACTIVITIES IN CELL FRACTIONS OF                                     METHYLOTROPHS                                                                          % Distribution in Cell Fraction                                                            Methane-Hydroxyl-                                                Propylene-Epoxidizing.sup.a                                                                ating.sup.a                                                      Activity     Activity                                                Microorganism                                                                            P(40)   P(80)   S(80)                                                                              P(40) P(80) S(80)                             ______________________________________                                        Type 1 Obligate                                                               Methylotrophs                                                                 Methylomonas sp.                                                              (CRL-17, NRRL                                                                            85      15      0    87    13    0                                 B-11,208)                                                                     Methylococcus                                                                 capsulatus 89      11      0    90    10    0                                 (Texas,ATCC                                                                   19,069)                                                                       Type II Obligate                                                              Methylotrophs                                                                 Methylosinus                                                                  sp. (CRL-15, NRRL                                                                        87      13      0    88    12    0                                 B-11,202)                                                                     Methylosinus                                                                  trichosporium                                                                            82      18      0    83    13    0                                 (CB3b, NRRL                                                                   B-11,196)                                                                     Faculative                                                                    Methylotroph                                                                  Methylobacterium                                                              sp.        85      15      0    82    18    0                                 (CRL-26, NRRL                                                                 B-11,222)                                                                     ______________________________________                                         .sup.a Reactions were carried out as described in Example 1. The product      of reaction was estimated by gas chromatography after 5, 10 and 15 min. o     incubation of reaction mixture at 30° C. on a rotary shaker.      

                  TABLE IX                                                        ______________________________________                                        THE RATE OF METHANE HYDROXYLATION -                                           AND PROPYLENE-EPOXIDATION IN THE CELL-                                        FRACTIONS OF METHYLOTROPHS                                                             Cell Fraction                                                                 Propylene-Oxidizing.sup.a                                                                  Methane-Oxidizing.sup.a                                          Activity     Activity                                                Microorganism                                                                            P(40)   P(80)   S(80)                                                                              P(40) P(80) S(80)                             ______________________________________                                        Type 1 Obligate                                                               Methylotroph                                                                  Methylomonas                                                                  sp. (CRL-17,                                                                             2.2     2.0     0    2.9   2.7   0                                 NRRL B-11,208)                                                                Methylococcus                                                                 capsulatus 2.6     2.0     0    3.8   3.9   0                                 (Texas, ATCC                                                                  19,069)                                                                       Type II Obligate                                                              Methylotroph                                                                  Methylosinus                                                                  sp. (CRL-15,                                                                             3.8     3.7     0    4.8   4.2   0                                 NRRL B-11,202)                                                                Methylosinus                                                                  trichosporium                                                                            2.8     2.5     0    3.1   3.0   0                                 (OB3b, NRRL B-11,196)                                                         Faculative                                                                    Methylotroph                                                                  Methylo-                                                                      bacterium sp.                                                                            1.2     1.1     0    2.7   2.8   0                                 (CRL-26, NRRL                                                                 B-11,222)                                                                     ______________________________________                                         .sup.a Reactions were carried out as described in Example 1. The product      of the reaction was estimated by gas chromatography after 5, 10 and 15        min. of incubation of reaction mixtures at 30° C. on a rotary          shaker. The rate of oxidation is expressed as μ moles of product forme     per hr. per mg. of protein.                                              

The particulate fractions [P(40) and P(80)] from various organisms alsocatalyzed the epoxidation of other n-alkenes (ethylene, 1-butene, and1,3-butadiene) to the corresponding 1,2-epoxides and the hydroxylationof methane and ethane to the corresponding alcohols. Table X shows therace of oxidation of various n-alkanes and n-alkenes by the P(40)particulate fraction of Methylosinus sp. (CRL-15, NRRL B-11,202). Theproduct of oxidation was identified by gas chromatography afterincubating P(40) fraction with various substrates at 30° C. for 10 min.

                  TABLE X                                                         ______________________________________                                        OXIDATION OF n-ALKENES AND n-ALKANES BY P(40)                                 PARTICULATE FRACTION OF METHYLOSINUS SP.                                      (CRL-15, NRRL B-11,202)                                                                              Rate of Product Formation.sup.a                        Substrate                                                                              Product       (μ moles/hr/mg of protein)                          ______________________________________                                        Ethylene Ethylene Oxide                                                                              1.27                                                   Propylene                                                                              Propylene Oxide                                                                             4.1                                                    1-Butene Epoxy butane  2.18                                                   Butadiene                                                                              Epoxy butene  0.63                                                   1-Pentene                                                                                --          0                                                      Methane  Methanol      4.8                                                    Ethane   Ethanol       3.2                                                    ______________________________________                                         .sup.a Reactions were carried out as described in Example 1. The product      of the reaction was estimated by gas chromatography after 5, 10, and 15       min. of incubation of reaction mixture at 30° C. on a rotary           shaker.                                                                  

Methylosinus sp. (CRL-15, NRRL B-11,202) was selected for furtherstudies on the influence of various environmental factors on themethane- and propylene- oxidizing activities in cell-free systems.

Effect of Particulate Fraction Concentration

The effect of the P(40) particulate fraction concentration on thehydroxylation of methane and epoxidation of propylene was examined. Theproduction of methanol and propylene oxide was directly dependent uponthe concentration of particulate fraction ranging from 1-6 mg. ofprotein per ml. The rate of reaction was decreased upon furtherincreasing the particulate protein concentration to 8 mg./ml.

Time Course of Reactions

The rate of formation of methanol and propylene oxide by hydroxylationof methane and epoxidation of propylene respectively, by the P(40)particulate fraction of Methylosinus sp. (CRL-15, NRRL B-11,202) waslinear with time up to 15 minutes.

Effect of pH

The effect of pH on the hydroxylation of methane and epoxidation ofpropylene by the P(40) particulate fraction of Methylosinus sp. (CRL-15,NRRL B-11,202) was examined by estimating the amount of methanol andpropylene oxide formed after 10 min. incubation of reaction mixtures.The optimum pH for both hydroxylation of methane and epoxidation ofpropylene was found to be 7.0. In carrying out these tests the reactionswere carried out as described in Example 1. The product of reaction wasestimated by gas chromatography after 5, 10 and 15 minutes of incubationof reaction mixture at 30° C. on a rotary shaker. 100% activity equals4.8 and 4.1 μmoles of methanol or propylene oxide formed respectively,per hour, per mg protein.

Effect of Temperature

The effect of temperature on the production of methanol and propyleneoxide by the P(40) particulate fraction of Methylosinus sp. (CRL-15,NRRL B-11,202) was examined after incubation of reaction mixtures for 10min. at various temperatures. The optimum temperature for epoxidation ofpropylene and hydroxylation of methane was found to be 35° C. Incarrying out these tests the reactions were carried out as described inExample 1. The product of reaction was estimated by gas chromatographyafter 5, 10 and 15 minutes of incubation of reaction mixture at 30° C.on a rotary shaker. 100% activity equals 5.0 and 4.2 μmoles of methanoland propylene oxide formed respectively, per hour per mg of protein.

Effect of Storage

It was noted that both the activity for hydroxylation of methane and theepoxidation of propylene by the P(40) particulate fraction ofMethylosinus sp. (CRL-15, NRRL B-11,202) decreased simultaneously whenstored at refrigerator (0°-4° C.) temperature. In carrying out thesetests the reactions were carried out as described in Example 1. Theproduct of reaction was estimated by gas chromatography after 5, 10 and15 minutes incubation of the reaction mixture at 30° C. on a rotaryshaker. 100% activity equals 4.8 and 4.1 μmoles of methanol andpropylene oxide formed respectively per hr. per mg. of protein.

Effect of Inhibitors

It has been reported that the oxidation of methane by cell suspensionsof methane-utilizing bacteria was inhibited by various metal-binding ormetal-chelating agents (Patel et al., J. Bacteriol. 126: 1017-1019(1976)). Hence, the effect of inhibitors on methane- andpropylene-oxidizing activities by the P(40) particulate fraction ofMethylosinus sp. (CRL-15, NRRL B-11,202) was examined. The production ofmethanol and propylene oxide was inhibited by various metal-bindingcompounds with different ligand combinations, i.e., nitrogen-nitrogen(α,α-bipyridyl), oxygen-nitrogen (8-hydroxyquinoline) andsulfur-nitrogen (thiourea, thiosemicarbazide) as shown in Table XI. Thissuggests the involvement of metal ion(s) in the oxidation of bothhydroxylation of methane, and epoxidation of propylene. Similarly, asshown in Table XIa these compounds also inhibit the hydroxylation ofmethane and epoxidation of propylene when using cell-containing enzymepreparations.

                  TABLE XI                                                        ______________________________________                                        EFFECT OF INHIBITOR ON THE ACTIVITY FOR                                       EPOXIDATION OF PROPYLENE AND HYDROXYLATION                                    OF METHANE BY METHYLOSINUS SP.                                                (CRL-15, NRRL B-11,202)                                                                        % Inhibition.sup.a                                                                  Propylene-                                                                              Methane-                                                 Concentra- Epoxidizing                                                                             Hydroxylating                                Inhibitor   tion (M)   Activity  Activity                                     ______________________________________                                        Control     --         0         0                                            α,α -Bipyridyl                                                                10.sup.-3  98        99                                           1,10-Phenanthroline                                                                       10.sup.-3  93        90                                           Potassium cyanide                                                                         10.sup.-3  98        100                                          Thiosemicarbazide                                                                         10.sup.-3  97        100                                          Thiourea    10.sup.-3  98        98                                           8-Hydroxyquinoline                                                                        10.sup.-3  75        80                                           ______________________________________                                         .sup.a Reactions were carried out as described in Example 1. The product      of the reaction was estimated by gas chromatography after 5, 10, and 15       min. incubation of the reaction mixture at 30° C. on a rotary          shaker. The uninhibited rates of methane and propylene oxidation were 4.5     and 4.1 μmoles of methanol and propylene oxide formed, respectively,       per hr. per mg. of protein in P(40) fraction of Methylosinus sp.              (CRL15,NRRL B11,202).                                                    

Effect of Metals

Since the methane mono-oxygenase from methane-utilizing bacteria is acopper or iron-containing protein (Tonge et al., J. Biochem., 161:333-344 (1977)), we have examined the effect of copper and iron salts onthe oxidation of methane and propylene by the P(40) particulate fractionof Methylosinus sp. (CRL-15, NRRL B-11,202). The rate of hydroxylationof methane to methanol and epoxidation of propylene to propylene oxidewas increased twofold in the presence of added copper salts (Table XII).

                                      TABLE XI.sup.a                              __________________________________________________________________________    Effect of Inhibitors on the Epoxidation of Propylene and the                  Hydroxylation of Methane                                                                % Inhibition                                                                  Methylosinus   Methylococcus  Methylobacterium                                trichosporium OB3b capsulatus                                                                organophilum                                                   (NRRL B-11,196)                                                                              (CRL Ml, NRRL B-11,219)                                                                      (CRL 26, NRRL B-11,222)               Inhibitor Epoxidation                                                                          Hydroxylation                                                                         Epoxidation                                                                          Hydroxylation                                                                         Epoxidation                                                                          Hydroxylation                  __________________________________________________________________________    Thiourea  100    100     100    100     100    100                            1,10-phenathroline                                                                      90     92      95     95      90     90                             α,60 -Bipyridyl                                                                   100    90      100    100     100    100                            Imidazole 95     90      95     95      100    100                            Potassium cyanide                                                                       100    100     100    100     95     95                             __________________________________________________________________________     The reactions were conducted as described in Example 1. The products were     estimated by gas chromatography after 1 hour of incubation at 30°      C. Each inhibitor was added at a final concentration of 1 mM.            

                                      TABLE XII                                   __________________________________________________________________________    EFFECT OF METALS ON THE ACTIVITY FOR                                          EPOXIDIZING PROPYLENE AND HYDROXYLATING                                       METHANE BY METHYLOSINUS SP. (CRL-15, NRRL B-11,202)                                            Propylene-Oxidizing.sup.a                                                                 Methane-Oxidizing.sup.a                                   Concentration                                                                         Activity    Activity                                         Metal    (M)     (μmoles/hr/mg protein)                                                                 (μmoles/hr/mg protein)                        __________________________________________________________________________    Control  --      4.5         4.0                                              Ferric Chloride                                                                        10.sup.-3                                                                             5.8         4.8                                              Ferrous Sulfate                                                                        10.sup.-3                                                                             5.9         4.8                                              Cuprous Chloride                                                                       10.sup.-3                                                                             9.2         7.2                                              Cupric Sulfate                                                                         10.sup.-3                                                                             9.0         7.1                                              __________________________________________________________________________     .sup.a Reactions were carried out as described in Example 1. The product      of the reaction was estimated by gas chromatography after 5, 10 and 15        min. of incubation of the reaction mixture at 30° C. on a rotary       shaker. The rates of oxidation were expresed as μmoles of product          formed per hr. per mg. of protein in P(40) fraction of Methylosinus sp.       (CRL15, NRRL B11,202).                                                   

Substrate Competition Experiments

The hydroxylation of methane and the epoxidation of propylene byparticulate fractions of methane-utilizing bacteria required oxygen andNADH. The question of whether the same or a similar enzyme was involvedin the oxidation of both substrates was examined by substratecompetition experiments. The experiment consisted of determining theeffect of methane on the oxidation of propylene to propylene oxide bythe P(40) particulate fraction of Methylosinus sp. (CRL-15, NRRLB-11,202). As shown in Table XIII, there was a reduction in the amountof propylene oxide formed in the presence of methane. Hence, methaneinhibited the conversion of propylene to propylene oxide, presumably bycompeting for the available enzymatic site.

                  TABLE XIII                                                      ______________________________________                                        EFFECT OF METHANE ON PROPYLENE EPOXIDIZING                                    ACTIVITY BY P(40) PARTICULATE FRACTION OF                                     METHYLOSINUS SP. (CRL-15, NRRL B-11,202)                                                       Propylene Oxide Produced.sup.a                               Substrate        μmoles/hr/mg. protein                                     ______________________________________                                        Propylene        4.3                                                          Propylene + Methane                                                                            1.8                                                          (1:1, v/v)                                                                    Methane          0                                                            ______________________________________                                         .sup.a Reactions were carried out as described in Example 1. The product      of the reaction was estimated by gas chromatography after 5, 10 and 15        min. of incubation of the reaction mixture at 30° C. on a rotary       shaker.                                                                  

Similarly, methane affects the epoxidation of propylene fromcell-suspensions of methane-grown Methylosinus trichosporium OB3b (NRRLB-11,196) as shown in Table XIIIa.

                  TABLE XIII.sup.a                                                ______________________________________                                        EFFECT OF METHANE ON THE EPOXIDATION OF                                       PROPYLENE.sup.a                                                               Composition of   Propylene Oxide                                                                             %                                              Gaseous Phase    Formed (μmoles)                                                                          Inhibition                                     ______________________________________                                        Propylene + Helium + O.sub.2                                                                   1.6           0                                              (25:25:50 v/v)                                                                Propylene + Methane + O.sub.2                                                                  0.8           50                                             (25:25:50 v/v)                                                                ______________________________________                                         .sup.a The reactions were conducted as described in Example 1 except that     various gaseous compositions were used to maintain a constant propylene       partial pressure. Cellsuspensions of methanegrown Methylosinus                trichosporium OB3b (NRRL B11,196) (3.6 mg.) were used. Propylene oxide wa     estimated by gas chromatography after 15 minutes of the incubation.      

Stoichiometry of Propylene and Methane Oxidation

The particulate P(40) fraction of Methylosinus sp. (CRL-15, NRRLB-11,202) was used to determine the stoichiometry of hydroxylation andepoxidation reactions. The stoichiometry of methane orpropylene-dependant NADH oxidation, oxygen consumption and productformation was approximately 1:1:1 Table XIV. This is consistent withmethane or propylene oxygenation being catalyzed by a mono-oxygenase.

                  TABLE XIV                                                       ______________________________________                                        STOICHIOMETRY OF PROPYLENE EPOXIDATION                                        AND METHANE HYDROXYLATION BY P(40)                                            PARTICULATE FRACTION OF METHYLOSINUS SP.                                      (CRL-15, NRRL B-11,202).sup.a                                                 Substrate                                                                             Product Formed                                                                             NADH Oxidized                                                                              O.sub.2 Consumed                            (μmoles)                                                                           (μmoles)  (μmoles)  (μmoles)                                 ______________________________________                                        Propylene                                                                             Propylene Oxide                                                       5.0     4.5          5.0          4.8                                         Methane Methanol                                                              5.0     4.2          4.8          4.6                                         ______________________________________                                         .sup.a Under identical condition of reaction, the estimation of NADH          oxidized was carried out spectrophotometrically, the estimation of oxygen     consumed was measured polarographically, and estimation of product formed     was carried out by gas chromatography.                                   

As a comparison the stoichiometry of the epoxidation of propylene by acell-suspension of Methylosinus trichosporium OB3b (NRRL B-11,196) wasdetermined as follows. The reaction mixture (3.0 ml.) contained 0.05 Msodium phosphate buffer, pH 7.0 and 3.6 μmoles of propylene. Thereaction was initiated by the injection of 0.1 ml. of cell-suspension(3.1 mg. protein). A correction was made for the endogenous consumptionof oxygen. The amount of oxygen consumed during the reaction (3 min.)was determined polarographically with a Clark oxygen electrode. Thepropylene consumed and the propylene oxide formed was estimated by gaschromatography. The propylene consumed was 0.29 μmoles, the oxygenconsumed was 0.30 μmoles and the propylene oxide formed was 0.28 μmoles.

To further demonstrate that the enzyme activity is in the particulatefraction (not in the supernatant) the following experiments were carriedout. Cells of methane-grown Methylococcus capsulatus (CRL M1, NRRLB-11,219) were obtained by the method of Example 1. The crude extractafter 10,000×g. centrifugation of sonically disrupted (3×50 sec., WaveEnergy Ultrasonic Oscillator, Model W 201) was found to have no activityfor either epoxidation or hydroxylation. However, when the cells weredisrupted by passing twice through a French pressure cell (1000 Kg.pressure), both activities were found in the crude extract after10,000×g. centrifugation. All of the activity in the crude extract wascollected as a particulate fraction by further centrifugation of thecrude extract as 40,000×g. for 90 min. at 4° C. NADH stimulated both theepoxidation and the hydroxylation reactions as shown in Table XV.

                  TABLE XV                                                        ______________________________________                                        EPOXIDATION AND HYDROXYLATION ACTIVITIES                                      IN CELL-FREE FRACTION OF                                                      METHYLOCOCCUS CAPSULATUS (CRL M1, NRRL B-11,219).sup.a                                         Oxidation Rate                                                               (nmoles)/30 min/assay)                                                          Epoxidation                                                                              Hydroxylation                                                      of         of                                               Cell-Free Fractions                                                                             Propylene  Methane                                          ______________________________________                                        (1)  Particulate fraction                                                                           750        500                                               (10,000 × g.- 40,000 × g.)                                   (1)  + NADH           900        650                                          (2)  Supernatant fraction                                                                           0          0                                                 of 40,000 × g.                                                     (2)  + NADH           0          0                                            ______________________________________                                         .sup.a The cells were disrupted by French Press as described above. NADH      (2.5 μmoles) was added into the reaction mixture where indicated. The      amount of protein in the particulate fraction and the 40,000 × g.       supernatant fraction used was 1 mg. and 2.5 mg., respectively. Each assay     contained 0.5 ml. reaction mixture.                                      

SUMMARY OF EPOXIDATION SYSTEM

Both the system of Pseudomonas aeruginosa demonstrated by Van derLinden, Biochim. Biophys. Acta., 77:157-159 (1963) and the system ofPseudomonas oleovorans, Abbott and Hou, Appl Microbiol., 26: 86-91(1973) epoxidized liquid 1-alkenes from C₆ to C₁₂, but not gaseousalkenes.

The present invention provides for the epoxidation of ethylene,propylene, 1-butene and butadiene by cell suspensions of all threedistinct groups of methane-utilizing bacteria. The epoxidation ofalkenes and the hydroxylation of methane were not found under anaerobicconditions or in methanol-grown cells, suggesting that the enzyme systemis inducible. The product 1,2-epoxides accumulated extracellularly. Thenonenzymic degradation of propylene oxide in the assay system disclosedwas not significant even after a prolonged incubation time. Van derLinden, supra, demonstrated the production of 1,2-epoxyoctane from1-octene by heptane-grown cells of Pseudomonas sp. and also stated thatthe epoxide was not further oxidized enzymatically. However, May andAbbott, Biochem. Biophys. Res. Commun., 48:1230-1234 (1972) and J. Biol.Chem., 248: 1725-1730 (1973) reported that when 1-octene was supplied asa substrate to the ω-hydroxylation enzyme system of P. oleovorans, both8-hydroxyl-1-octene and 1,2-epoxyoctane were formed. In addition, Abbottand Hou, supra, found that the methyl group of the latter compound wasalso susceptible to hydroxylation. The present results obtained from thestudies of viable cell suspensions of the methane-utilizing bacteria,however, indicated that propylene oxide was not further metabolizedenzymatically.

Van der Linden, supra, showed that the epoxide accumulation from1-octene by Pseudomonas aeruginosa was accompanied by the metabolism ofa large quantity of 1-octene via methyl group epoxidation. In theepoxidation of propylene by cell suspensions of methane-utilizingbacteria, however, no formation of 3-hydroxy propene-1 was detected.

Both the epoxidation of the C₂ -C₄ 1-alkenes and the hydroxylation ofmethane with the cell suspensions were inhibited by variousmetal-binding and metal-chelating agents, indicating the involvement ofmetal(s)-containing enzyme system(s). The similar extent of inhibitionfor both propylene and methane oxidation (Table XIa) indicated that theepoxidation and hydroxylation reaction may be catalyzed by the same or asimilar enzyme system. The epoxidation of propylene to propylene oxideby a cell suspension of methane-grown strain Methylococcus capsulatusNRRL B-11,219 was inhibited (50%) in the presence of the hydroxylationsubstrate, methane (Table XV). This clearly suggests a comparisonbetween the hydroxylation substrate and the epoxidation substrate for asingle enzyme system. It is likely that the methane mono-oxygenaseenzyme system catalyzes both the epoxidation of alkene and thehydroxylation of methane. May and Abbott publications, supra, havereported that the ω-hydroxylation system from Pseudomonas oleovoranscatalyzed both the epoxidation of 1-octene and the hydroxylation ofn-octane.

The optimum conditions for the in vivo epoxidation of propylene by cellsuspensions of the three distinct groups of methane-utilizing bacteriaare quite similar. The pH optima were around 6-7 and the temperatureoptimum was around 35° C. The apparent decrease in epoxidation above 40°C. may be due to both the instability of the mono-oxygenase system andthe volatility of the product propylene oxide. (b.p. 35° C.).

Both the hydroxylation and epoxidation activities are located in thecell-free particulate fraction precipitated between 10,000×g. and80,000×g. centrifugation. Tonge et al., Biochem. J., 161: 333-344 (1977)and FEBS Lett., 58: 293-299 (1975) have reported the purification of amembrane-bound methane mono-oxygenase from the particulate fraction(sedimented between 10,000×g. and 150,000×g. centrifugation) ofMethylosinus trichosporium OB3b. Recently, but subsequent to ourdiscoveries Colby et al., Biochem. J., 165: 395-402 (1977) demonstrateda unique soluble methane monooxygenase from Methylococcus capsulatus(Bath strain) which catalyzes the oxidation of n-alkanes, n-alkenes,ethers and alicyclic, aromatic and heterocyclic compounds. The strainsfrom the three distinct groups of methane-utilizing bacteria that wehave examined all catalyze the epoxidation of gaseous alkenes (C₂ -C₄)and the hydroxylation of gaseous alkanes (C₁ -C₄). Also, we unexpectedlyfound the enzyme activity is in the particulate fraction (i.e., thematerial which sediments when the supernatant after centrifuging brokencells at 10,000×g. for 30 minutes is centrifuged for 1 hour at 10,000×g.or greater), not the soluble fraction (i.e., the supernatant aftercentrifuging broken cells at 80,000×g. or greater for 1 hour.

Differential centrifugation of broken-cell suspensions of Methylomonassp. (CRL-17, NRRL B-11,208) and Methylococcus capsulatus (Texas ATCC19,069), (Type I obligate methlotrophs); Methylosinus sp. (CRL-15, NRRLB-11,202) and Methylosinus trichosporium (OB3b, NRRL B-11,196) (Type IIobligate methylotrophs); and Methylobacterium sp. CRL-26, NRRL B-11,222)(a facultative methylotroph) has yielded cell-free particulate fractionsthat catalyzed the hydroxylation of n-alkanes and the epoxidation ofn-alkenes. Both activities mainly resided in the P(40) fraction and weredependent upon the presence of oxygen, as well as electron carrier,e.g., NADH.

The hydroxylation of methane to methanol and the epoxidation ofpropylene to propylene oxide catalyzed by the P(40) particulate fractionof Methylosinus sp. (CRL-15, NRRL B-11,202) have similar pH andtemperature optima. Both activities were lost simultaneously duringstorage of the P(40) particulate fraction at refrigerator temperature.

The hydroxylation of methane and the epoxidation of propylene with thecell-free extracts were strongly inhibited by various metal-binding ormetal-chelating agents (Table XI). The rate of both reactions wereincreased twofold in the presence of copper or iron salts (Table XII).This suggests the involvement of a metal-containing enzyme system in theoxidation of both substrate. These results, and the stoichiometry of thehydroxylation and the epoxidation reactions indicate that both reactionsmay be catalyzed by the same metal-containing monooxygenase system. Thefact that conversion of propylene to propylene oxide was inhibited bymethane support this proposition.

It has been reported that the cell-free particulate fractions derivedfrom Methylococcus capsulatus (Texas) (Ribbons et al., J. Bacteriol.,122: 1351-1363 (1975)), Methylomonas methanica (Ferenci et al., J. Gen.Microbiol., 91: 79-91 (1975)) and Methylosinus trichosporium (OB3b)(Tonge et al., Biochem. J., 161: 333-344 (1977)) catalyzed oxygen- andNADH-dependent oxidation of methane, ethane, propane, butane, and carbonmonoxide. The oxidation of methane by particulate fractions of theseorganisms was inhibited by various metal-binding or metal-chelatingagents. However, epoxidation of n-alkenes was not reported for theseorganisms.

The methane monooxygenase from Methylosinus trichosporium (OB3b, NRRLB-11,196) has been purified and shown to be consisting of threecomponents: a soluble CO-binding cytochrome c, a copper-containingprotein (methane monooxygenase), and a small molecular weight protein(Tonge et al., 1977, supra).

In contrast to the above organisms, Colby et al., supra have reportedthe unique soluble methane monooxygenase activity from Methylococcuscapsulatus (Bath). The oxidation of methane by the soluble fraction ofthis organism was not inhibited by various metal-binding agents.Recently, Colby and Dalton (Biochem. J., 171: 461-468 (1978)) resolvedthe methane monooxygenase of Methylococcus capsulatus (Bath) into threecomponents and identified one of the components as an iron-containingflavoprotein.

The methane-oxidizing activities from the methylotrophic bacteriadescribed above is in the particulate fraction and different from thesoluble activity of Methylococcus capsulatus (Bath) disclosed by Colbyet al.

Van der Linden (1963, supra) demonstrated the production of 1,2-epoxidesfrom 1-octene by heptene-grown resting cells of Pseudomonas sp. Epoxideswere not detected as products of alkane metabolism and were not oxidizedby Pseudomonas sp. Thus, the role of epoxides in alkane metabolism isuncertain. Van der Linden postulated that the enzyme system that formsepoxides may be the same as the system that catalyzes the initialoxidation of alkanes. Cardini and Jurtshuk (J. Biol. Chem., 245:2789-2796 (1970)) found that a cell-free extract of a Corynebacteriumsp. carried out the oxidation of 1-octene to epoxyoctane in addition tohydroxylation of octane to octanol. McKenna and Coon (J. Biol. Chem.,245: 3883-3889 (1970)) isolated an enzyme system from Pseudomonasoleovorans that catalyzed the hydroxylation of n-alkanes (C₆ -C₁₂) andfatty acids. Subsequently, Abbott and Hou, supra and May and Abbott,supra reported that the enzyme system from Pseudomonas oleovorans alsocatalyzed the epoxidation of 1-alkenes in addition to the hydroxylationreactions. The enzyme systems from Pseudomonas and Corynebacterium sp.catalyzed epoxidation of C₆ -C₁₂ n-alkenes. Epoxidation of C₂ -C₅n-alkenes was not catalyzed by the Pseudomonas enzyme systems.

We have unexpectedly demonstrated that the three distinct groups ofmethane-oxidizing bacteria catalyze the hydroxylation of n-alkanes (C₁-C₄) as well as the epoxidation of n-alkenes (C₂ -C₄). Furthermore, thehydroxylation and the epoxidation reactions are catalyzed by the same ora similar NADH-dependent monooxygenase.

In addition to methylotrophic bacteria, other microorganisms can be usedto carry out the epoxidation of C₂ -C₄ alkenes. These include bacteria,fungi and yeast which grow on short chain alkanes. The methylotrophicbacteria (obligate or facultative) or the other microorganisms are growneither on methane as a sole source of carbon, or on another carboncompound (in the presence of methane or another inducer), and the cells,or enzymes derived therefrom, may be used in the process of the presentinvention.

ALCOHOL OXIDATION SYSTEMS

As shown and discussed above (Table IX) resting-cell suspensions ofmethane- and methanol-grown microbial cells oxidized (dehydrogenated) C₃-C₆ secondary alcohols to their corresponding methyl ketones. Theproduct methyl ketones accumulated extracellularly as determined byanalysis of the supernatant of the centrifuged reaction mixture. Controlexperiments with heat-killed cells indicated that the methyl ketoneswere produced enzymatically. In these tests, secondary alcoholdehydrogenase (SADH) activity was found in all of the C₁ -utilizerstested. Further tests have shown that SADH activity was found in cellsuspensions of methanol-grown or methylamine-grown microorganisms.However, the SADH does not appear to be a constitutive enzyme since theSADH enzyme activity was not found in succinate-grown facultative C₁-utilizers.

To prepare the cell-free secondary alcohol dehydrogenous (SADH) system,the washed cells were disrupted with a Wave Energy UltrasonicOscillator, Model W201 (Wave Energy System, Inc., Newtown, Pa.) andcentrifuged at 20,000×g. for 30 minutes. The clear supernatant containedthe SADH activity. The enzyme activity was measured with a fluorescencespectrophotometer (Perkin Elmer, Model MPF 44A) by following theformation of reduced NAD (EX 340 nm, Em 460 nm). The formation ofreduced NAD was also followed with an absorption spectrophotometer at340 nm. The assay system (3 ml.) contained: potassium phosphate bufferpH 7.0; 150 μmol.; NAD 1 μmol.; a given amount of enzyme preparation;and secondary alcohol 10 μmol. The reaction was started by the additionof substrate. One unit of enzyme activity represents the reduction ofone μmole NAD per minute. Protein concentrations were determined by theLowry method as referenced above.

The following summarizes tests conducted on the optimal conditions forthe production of methyl ketones from C₃ -C₆ sec. alcohols. It will beunderstood that these were the optimal conditions found and theinvention is not meant to be bound by them. Conversions can still beobtained by deviating from optimum indicated below, but with loweryields and conversions.

Time Course

The production of 2-butanone from 2-butanol reached a maximum after 14hours of incubation in batch experiments in all the microorganismstested. The amount of 2-butanone did not decline after 30 hours ofincubation. The rate of 2-butanone production was linear for the first 4hours. Therefore, the production of 2-butanone was measured within thisinterval whenever the effect of a variable was tested.

pH

The effect of pH on the production of 2-butanone was studied with tris(hydroxymethyl) amino methane-HCl buffer (0.05 M) for pH values of 8.0to 10.0, and 0.05 M potassium phosphate buffer for values from 5.0 to8.0. A pH around 8.0 was found to be the optimum for 2-butanoneformation in all the microorganisms tested. Of the new strains,Methylobacterium organophilum CRL 26 (NRRL B-11,222), showed highactivity at both 8 and 9. The yeast cells appeared less affected by pHin the production of 2-butanone.

Temperature

The temperature optimum for the production of 2-butanone bycell-suspensions was about 35° C. except for the yeast culture, whichhad an optimum of about 40° C.

Substrate Concentration

Various concentrations of 2-butanol were added to cell-suspensions ofyeast and of strain Pseudomonas sp. ATCC 21439. The production of2-butanone was assayed after 35 min. of incubation. The amount of2-butanone produced was dependent on the amount of substrate initiallyadded. A 2-butanol concentration of about 50 μmoles supported maximum2-butanone production.

Cell Concentration

The cell concentration also has an influence on the rate of 2-butanoneproduction. The amount of 2-butanone accumulated after 2 hours ofincubation increased linearly as the cell concentration was increased byto about 12 mg./0.5 ml. for yeast and for Methylococcus capsulatus CRLM1 (NRRL B-11,219) and about 17 mg./0.5 ml. for strains Methylobacteriumorganophilum CRL 26R₆ (NRRL B-11,222), Methylosinus trichosporium OB3b(NRRL B-11,196) and Pseudomonas sp. ATCC 21.439.

Product Inhibition and Further Oxidation

Examination of the time course of 2-butanone production revealed thatthe rate decreased after 4 hours of incubation, suggesting, among otherpossibilities, either product inhibition or further oxidation of2-butanone. To test these possibilities, 8 μmoles of 2-butanone wasadded to viable or heat-killed cell suspensions and incubated under theconditions described above for producing the 2-butanone. No decline wasobserved in 2-butanone concentration in all the heat-killed cellsuspensions, but 2-butanone slowly disappeared in the presence of viablecells of all the strains tested. When 2-butanol (5 μl/0.5 ml. reactionmixture) was added to viable cell-suspensions along with the exogenouslysupplied 2-butanone, a net increase in 2-butanone production wasdetected. The reaction rates were identical to those where the secondaryalcohol was initially converted to the methyl ketone and were notaffected by the presence of the exogenously supplied 2-butanone. Thesedata indicate that there is no product inhibition in the production of2-butanone. A small amount of further oxidation of 2-butanone by viablecell-suspensions was observed. The decrease in 2-butanone productionrate after 4 hours of incubation may be due to the depletion of otherrequirement(s), e.g., a cofactor(s).

Inhibition Studies

The production of 2-butanone from 2-butanol by cell suspensions of thestrains tested was inhibited by metal-chelating agents such as 1,10-phenanthroline and α,α-dipyridyl. However, the activity was notinhibited by sodium cyanide or thiourea which suggests metal involvementfor the enzyme. The results of the inhibition tests are shown in TableXVI.

                  TABLE XV                                                        ______________________________________                                        EFFECT OF METAL-CHELATING AGENTS AND OTHER                                    INHIBITORS ON THE PRODUCTION OF 2-BUTANONE BY                                 CELL SUSPENSIONS OF METHANOL-GROWN                                            METHYLOCOCCUS CAPSULATESCRL M1 (NRRL B-11,219)                                Metal-Chelating Agents                                                                       Concentration                                                                              Inhibition (%)                                    ______________________________________                                        Sodium cyanide 1 mM         0                                                 Sodium azide   1 mM         10                                                EDTA           1 mM         70                                                1,10-phananthroline                                                                          1 mM         95                                                α,α-bipyridyl                                                                    1 mM         75                                                Thiourea       1 mM         0                                                 ______________________________________                                    

Substrate Specificity

The substrate specificity for the oxidation of C₃ -C₆ secondary alcoholsby the strains of C₁ -utilizers was studied. Among the secondaryalcohols, 2-propanol and 2-butanol were oxidized at higher rates;2-pentanol, 2-hexanol, and 2-heptanol were oxidized at a much slowerrate. The oxidation products of these secondary alcohols were thecorresponding methyl ketones, as determined by GC retention timecomparisons with authentic standards.

Cell-Free System

Cell-free soluble extracts from sonically disrupted cells of new strainsand known strains also oxidized 2-butanol to 2-butanone. These resultsare shown in Table XVII. However, all of the cell-free systems testedrequired the addition of a cofactor, NAD, for its activity. Othercofactors tested (including NAD(P)H, NADP, phenazine methosulfate, GSH,FAD, potassium ferricyanide, and dichlorophenol indophenol) were noteffective. The stoichiometry for the consumption of 2-butanol, thereduction of NAD, and the formation of 2-butanone was obtained forPseudomonas sp. ATCC 21,439 as shown in Table XVIII. This is the firstreport of an NAD-dependent secondary alcohol dehydrogenase.

The experimental procedure for the tests reported in Table XVII were asfollows: 1 mg protein of crude extract was added into a 0.5 ml. 0.05 Mphosphate buffer (pH 7.0) in a 10-ml. vial. One μmol NAD and 10 μmol2-butanol was added, and the vial was sealed with a rubber cap tominimize evaporation. The reaction mixture was incubated at 30° C. on awater bath. A 3 μl sample was removed with a syringe at 15 min. ofincubation and was assayed with g.l.c. Catalytic activity was alsoassayed by fluorescence spectrophotometry. Data obtained from bothg.l.c. and fluorescence spectrophotometric assays agreed with eachother. Comparable conversions (as reported in Table XXVII) for extractsderived from CH₃ NH₂ and HCOOCH₃ grown microbes were also obtained.

                  TABLE XVII                                                      ______________________________________                                        OXIDATION OF 2-BUTANOL TO 2-BUTANONE BY CELL-                                 FREE SOLUBLE EXTRACTS OF C1-UTILIZING                                         MICROBES                                                                                                    Conversion                                                                    Rate                                                                 Growth   (nmoles/min/                                    Microbes             Substrate                                                                              mg protein)                                     ______________________________________                                        Obligate methylotrophs                                                        Type I membrane structure                                                     Methylosinus trichosporium OB3b                                                                    CH.sub.4 4.5                                             (NRRL B-11,196)                                                               Methylosinus trichosporium OB3b                                                                    CH.sub.3 OH                                                                            2.4                                             (NRRL B-11,196)                                                               Type II membrane structure                                                    Methylococcus capsulatus CRL M1                                                                    CH.sub.4 3.2                                             (NRRL B-11,219)                                                               Methylococcus capsulatus CRL M1                                                                    CH.sub.3 OH                                                                            2.0                                             (NRRL B-11,219)                                                               Facultative methylotroph                                                      Methylobacterium organophilium CRL 26                                                              CH.sub.4 1.8                                             (NRRL B 11,222)                                                               Methylobacterium organophilium CRL 26                                                              CH.sub.3 OH                                                                            2.5                                             (NRRL B 11,222)                                                               Obligate methanol-utilizer                                                    Pseudomonas sp. CRL 75 ATCC 21439                                                                  CH.sub.3 OH                                                                            25.0                                            Yeast                                                                         Hansenula polymorpha ATCC 26012                                                                    CH.sub.3 OH                                                                            23.2                                            ______________________________________                                         .sup.a Cells were disrupted as described and the supernatant of 10,000        × g. centrifugation was used for the enzyme assay.                 

                  TABLE XVIII                                                     ______________________________________                                        STOICHIOMETRY OF THE PRODUCTION OF                                            2-BUTANONE FROM 2-BUTANOL BY CELL-FREE                                        EXTRACTS OF STAIN ATCC-21439                                                                        NAD                                                     Experi-                                                                             2-Butanol Consumed                                                                            Consumed.sup.b                                                                          2-Butanone.sup.a                              ment  (nmoles)        (nmole)   Produced (nmole)                              ______________________________________                                        1     260             270       250                                           2     530             540        20                                           ______________________________________                                         The reaction mixtures 3 ml (1.0 mg protein) were incubated at 30°      C. for 10 min. (exp. 1) and for 20 min. (exp. 2) in the presence of 1.0       μmoles NAD and 10 μmoles 2butanol.                                      .sup.a Determined gas chromatographically.                                    .sup.b Determined fluorescence spectrophotometrically. Endogenous             consumption of NAD was corrected.                                        

Purification and Properties of Secondary Alcohol Dehydrogenase

Secondary alcohol dehydrogenase (SADH) from an obligate methanolutilizer, Pseudomonas sp. ATCC 21439 was purified as follows. The cellswhich had been grown on methanol as the carbon source as described inthe preceding examples were suspended in 300 ml. 0.05 M sodium phosphatebuffer, pH 7.0 with 0.5 mM dithiothretol (buffer A) and were disruptedsonically (5×1 min.). The crude extract was separated by centrifugation.The crude extract was heat-treated at 50° C. in a water bath for 10minutes. The resulting precipitate was removed by centrifugation. To thesupernatant solution, 25 ml of protamine sulfate solution (2% solutionin 0.1 M Tris base) was added dropwise with continuous stirring. Afterstanding for 30 minutes, the extract was centrifuged. The supernatantsolution was fractionated with solid ammonium sulfate. The materialprecipitating between 30 and 60% saturation was collected and wasdialized overnight against buffer A. The The dialized material wasapplied to a DEAE-cellulose column (3 cm by 35 cm) that had beenequilibrated with buffer A. The secondary alcohol dehydrogenase activitywas eluted in the void volume. This DEAE-cellulose eluate wasconcentrated by ammonium sulfate fractionation. Material precipitatingbetween 30 and 50% ammonium sulfate saturation was collected bycentrifugation and dialyzed overnight against A. This fraction wasfurther washed and filtered through an Amicon unit with XM 50 membrane.The concentrated fraction (6 ml) inside the Amicon unit was applied toan Affi-Gel Blue column (0.8 cm×18 cm) which had been equilibrated withbuffer A for affinity chromatography. The column was washed overnightwith buffer A (0.18 ml./min.) and then was eluted with buffer Acontaining 5 mM NAD. Each 1 ml. fraction was collected. SADH activitywas located in tube numbers 8-12. A summary of the purification steps isshown in Table XIX.

                  TABLE XIX                                                       ______________________________________                                        PURIFICATION OF SECONDARY ALCOHOL                                             DEHYDROGENASE FROM PSEUDOMONAS SP.                                            ATCC 21439                                                                               Vol-    Pro-   Sp. Act.                                                       ume     tein   (units/mg                                                                              Total Yield                                Procedures (ml)    (mg)   protein  units (%)                                  ______________________________________                                        Crude extract                                                                            250     2698   25       67450 100                                  Heat treatment                                                                           245     949    67.5     64080 95                                   Protamine sulfate                                                                        260     526    103.8    54640 81                                   (NH.sub.4).sub.2 SO.sub.4                                                                30      232    200      46450 69                                   (30-60% sat.)                                                                 DEAE-cellulose                                                                           150     42.2   875      37160 55                                   column                                                                        Amicon filtration                                                                        6       22.0   1,500    33050 49                                   (XM-50)                                                                       Affi-Gel Blue                                                                            5       0.34   65,600   22300 33                                   column                                                                        ______________________________________                                    

The purified secondary alcohol dehydrogenase enzyme (SADH) may be useddirectly for converting C₃ -C₆ secondary alcohols to the correspondingmethyl ketones by the procedures described above; however, a source ofNAD⁺ must be added to the reaction medium. One can determine theNAD-linked secondary alcohol dehydrogenase activity with a fluorescencespectrophotometer (Perkin Elmer, Model MPF 44A) by following theformation of reduced NAD (Ex 340 nm, Em 460 nm). The assay system (3ml.) will typically contain: sodium phosphate buffer pH 7.0, 150 μmol;NAD 1.0 μmol; a given amount of enzyme preparation; and 20 μmolsecondary alcohol. The reaction is started by the addition of secondaryalcohol. One unit of SADH enzyme activity represents the reduction ofone nmole NAD per minute.

The purification procedure outlined in Table XIX may be modified byomitting the heat-treatment. A higher specific activity can be obtainedby omitting the heat-treatment (a specific activity of 45 units ofSADH/mg. protein from Pseudomonas sp. ATCC 21,439 was obtained). Thepresence of a reducing agent such as dithrothretol in the dialyzingbuffer was found essential during the dialysis of the materialprecipitated between 30-60% (NH₄)₂ SO₄ saturation. In one specificexperiment the Affi-Gel Blue column was scaled-up to a size of 2.5 cm×25cm. From 10 liters of crude extract containing 200 g. protein, a 45 mg.pure SADH fraction with specific activity of 65,600 SADH units/mg.protein (33% recovery) was obtained.

Metal analysis of the purified SADH enzymes were conducted by x-rayfluorescence technique with a Phillips PW 1220C semi-automatic vacuumspectrograph. In carrying out the metal analysis the purified SADH wasfirst washed thoroughly with deionized distilled water and then driedevenly on an Amicon XM 50 ultrafiltration membrane. This membrane wasthen assayed by x-ray fluorescence technique. Control experiments aretaken with blank ultrafiltration membranes. The minimum amount of metaldetectable qualitatively and quantitatively by this method are >0.02 μg.and >0.5 per cm², respectively. Metal analysis by this technique on thepurified bacteria derived SADH enzymes showed 0.7 μg. zinc/cm² of theultrafiltration membrane. This is equivalent to two moles of zinc permole of SADH enzyme, or one zinc per subunit. No other metal wasdetected.

The molecular weight of the purified SADH was determined by acrylamidegel electrophoresis using 7.5% gel and stained with both coomassiebrilliant blue and with nitro-blue tetrazolium activity stain. Sodiumdodecyl sulfate-gel electrophoresis in a 10% gel system and thedissociation of enzyme protein were conducted using SDS-PAGE standards.Both the protein stain and the enzyme activity stain of the purifiedSADH enzymes tested showed a single protein band. The mobility on thegel electrophoresis of SADH from the distinct types of methanol-grownbacterial cells were identical. Yeast derived SADH had a faster mobilitytoward the anode on the gel electrophoresis. The molecular weights ofseveral bacterial and yeast derived and purified SADH enzymes each hadan identical molecular weight of 95,000 dalton as estimated by a Bio-Gelagarose A-1.5 column. SDS-gel electrophoresis of the purified enzymesshowed two identical subunits of 48,000 dalton.

The optimum pH and temperatures for activity of the purified SADH was8-9 and 30°-35° C., respectively, although wider ranges of pH andtemperatures did not significantly affect the enzyme activity. Theactivation energy for SADH, as calculated from the Arrhenius plots ofvelocity vs. the reciprocal of the absolute temperature, is 19.8 K cal.The absorption spectrum of the purified SADH fraction showed no peak inthe visible region.

The Michaelis constants (K_(m)) of SADH calculated from Line-Weaver-Burkplot was 1.1×10⁻⁵ M for NAD. Similar reaction rates were obtainedwhether SADH was preincubated for 10 min. with either NAD or 2-butanol.This indicates that the addition of substrates in the SADH reaction isnot an obligatory order and is rather a random mechanism. No consumptionof dissolved oxygen was observed during the reaction.

The effect of metal-chelating agents and thioreagents on the activity ofthe purified SADH enzyme were studied. The SADH activity was inhibitedas follows (% inhibition, activity measured spectrofluorometrically andeach inhibitor added at a final concentration of 1 mM): iodoacetic acid,0%; N-ethylmalemide, 6%; p-hydroxymercuribenzoate, 100%;5,5'-dithiobis(2-nitrobenzoic acid), 100%; sodium cyanide, 0%; sodiumazide, 10%; EDTA, 63%; 1,10-phenanthroline, 95%; α,α-bipyridyl, 70%;thiourea, 0%; cupric, 25%; ferric, 35%; ferrous, 50%; nickel, 20%; andZn⁺⁺, Co⁺⁺, Mn⁺⁺, or Mg⁺⁺, 0%. Despite the fact that SADH contains 2moles of zinc per mole of enzyme, the addition of exogenous zinc did notstimulate its activity. The possibility of ethanol or n-propanol as aninhibitor was studied. Despite their structural similarity in competingwith 2-butanol for the alkyl binding site(s), both of them did notinhibit SADH activity.

The substrate specificity of purified SADH was highest for 2-propanoland 2-butanol. It also oxidized at a lower rate, 2-pentanol, 2-hexanol,acetaldehyde, propanol, cyclohexanol, butane, 1,3-diol and butane2,3-diol. Primary alcohols were not substrates of purified SADH. Itappears that a hydrophobic carbon moiety adjacent to the secondaryalcohol is required for enzyme activity.

The purified SADH enzyme was analyzed for amino acids with a BeckmanModel 120B amino acid analyzer following acid hydrolysis of the enzyme.The results of the amino acid analysis are summarized in Table XX. Thevalues are expressed as average number of residues per molecule obtainedfrom 24, 48 and 72 hours acid hydrolysis, assuming a molecular weight of95,000. Only two residues of cysteine were detected.

                  TABLE XX                                                        ______________________________________                                        AMINO ACID COMPOSITION OF PURIFIED SADH.sup.a                                                    No. of Residues/                                           Amino Acid         95,000 dalton                                              ______________________________________                                        Lysine             52                                                         Histidine          14                                                         Arginine           26                                                         Cysteic Acid       2                                                          Aspartic Acid      78                                                         Threonine          26                                                         Serine             14                                                         Glutamine          76                                                         Proline            32                                                         Glycine            72                                                         Alanine            92                                                         Valine             68                                                         Methionine         6                                                          Isoleucine         54                                                         Leucine            74                                                         Tryosine           6                                                          Phenylalanine      28                                                         Tryptophane        28                                                         ______________________________________                                         .sup.a The secondary alcohol dehydrogenase enzyme was purified from cells     derived from Pseudomonas sp. ATCC 21,439 aerobicaly grown on methanol.   

Yeast Derived SADH

As previously indicated both cell suspensions and cell-free extracts ofC₁ -compound grown yeasts enzymatically convert C₃ -C₆ secondaryalcohols to the corresponding methyl ketones. Further, we specificallyfound that cell suspensions of the yeasts: Candida utilis ATCC 26,387;Hansenula polymorpha ATCC 26,012; Pichia sp. NRRL Y-11,328; Torulopsissp. strain A₁ NRRL Y-11,419; and Kloeckera sp. strain A₂ NRRL Y-11,420grown on various C₁ compounds (e.g., methanol, methylamine,methylformate), ethanol and propylamine catalyzed the oxidation of C₃-C₆ secondary alcohols to the corresponding methyl ketones. Cell-freeextracts of these yeasts catalyzed the NAD⁺ -dependent oxidation of theC₃ -C₆ secondary alcohols to the corresponding methyl ketones. Thepresence of NAD⁺ as an electron acceptor was essential in the case ofthe cell-free extract of these yeast derived enzymes. Primary alcoholswere not oxidized by this purified enzyme. The molecular weight of thepurified yeast derived SADH enzyme was 98,000 dalton as determined bygel filtration and the subunit size as determined by sodium dodecylsulfate gel electrophoresis was 48,000.

It is to be noted that the molecular weight of the purified SADH whetheryeast or bacteria derived is about 95,000, a determined by gelelectrophoresis, but the value may vary ±3,000 due to purificationprocedures used and experimental error.

The activity of the purified yeast derived SADH was inhibited bysulfhydryl inhibitors and metal-binding agents. The optimum pH of thepurified enzyme was determined to be about 8.

A typical yeast derived SADH enzyme was prepared as follows:

The yeasts were grown at 30° C. in 2.8 liter flasks containing 700 ml.mineral salts medium (described below) with 0.1% yeast extracts and0.4%, v/v methanol.

    ______________________________________                                        Yeast Growth Medium.sup.a                                                     ______________________________________                                        KH.sub.2 PO.sub.4     2.5 gm.                                                 NH.sub.4 NO.sub.3     2.5 gm.                                                 MgSO.sub.4 . 7H.sub.2 O                                                                             0.3 gm.                                                 KCl                   0.04 gm.                                                CaCl.sub.2            0.015 gm.                                               FeSO.sub.4 . 7H.sub.2 O                                                                             1.0 mg.                                                 CuSO.sub.4 . 5H.sub.2 O                                                                             0.01 mg.                                                H.sub.3 BO.sub.3      0.02 mg.                                                MnSO.sub.4 . 5H.sub.2 O                                                                             0.04 mg.                                                ZnSO.sub.4            0.14 mg.                                                MoO.sub.3             0.02 mg.                                                Yeast extract         1.0 gm.                                                 Methanol              4 ml.                                                   ______________________________________                                         .sup.a The followng composition is on a per liter basis.                 

The cells were harvested during exponential growth by centrifugation at12,000×g. for 15 min. The cell pellet was washed twice with 50 mMphosphate buffer, pH 7. The final pellet was resuspended in the samebuffer. Cell suspensions of yeasts grown on ethanol, methylamine, andmethylformate were prepared as described above using 0.4 v/v ethanol, 10mM methylamine and 10 mM methylformate as the sole source of carbon andenergy.

A 1 ml. aliquot of each washed cell suspension of yeasts grown onvarious carbon sources was put into 10 ml. vials at 40° C. Ten μl ofsecondary alcohol (2-propanol, 2-butanol, 2-pentanol and 2-hexanol) wasadded to the cell suspensions in an independent vial. The vials werethen incubated at 30° C. on a rotary water bath shaker at 200 rpm. Theproduct of oxidation of secondary alcohols was detected by gaschromatography retention time comparison and cochromatography withauthentic standard. As shown in Table XXI the cell suspensions of yeastscatalyze the conversion of isopropanol, 2-butanol, 2-pentanol, and2-hexanol to the corresponding methyl ketones. The products of oxidationof secondary alcohols were accumulated extracellularly and no furtheroxidation of products (methyl ketones) was revealed by gaschromatographic analysis.

                                      TABLE XXI                                   __________________________________________________________________________    OXIDATION OF SEC-ALCOHOLS TO KETONES                                          BY CELL SUSPENSIONS OF YEASTS.sup.a                                                             Conversion Rate                                                               (μmoles/hr/mg of protein)                                                  Isopropanol                                                                          2-Butanol                                                                           2-Pentanol                                                                           2-Hexanol                                         Growth  to     to    to     to                                      Organism  Substrate                                                                             Acetone                                                                              2-Butanone                                                                          2-Pentanone                                                                          2-Hexanone                              __________________________________________________________________________    Candida utilis                                                                          Methanol                                                                              6.2    6.8   1.5    0.8                                     ATCC 26387                                                                              Ethanol 5.2    5.2   1.0    0.72                                              Methylamine                                                                           5.0    5.0   1.2    0.61                                              Methylformate                                                                         5.6    6.2   1.3    0.75                                              Propylamine                                                                           4.2    4.2   0.9    0.52                                    Hansenula poly-                                                               morpha    Methanol                                                                              5.9    5.8   1.4    0.72                                    ATCC 26012                                                                              Ethanol 5.0    4.8   1.1    0.54                                              Methylamine                                                                           5.2    4.5   1.2    0.62                                              Methylformate                                                                         5.6    5.2   1.3    0.70                                              Propylamine                                                                           4.1    4.0   0.82   0.48                                    Pichia sp.                                                                              Methanol                                                                              5.2    6.8   1.2    0.50                                    NRRL-Y-11328                                                                            Ethanol 4.5    6.2   1.0    0.28                                              Methylamine                                                                           4.2    5.1   0.72   0.31                                              Methylformate                                                                         4.9    6.9   0.98   0.48                                              Propylamine                                                                           3.2    2.1   0.60   0.21                                    Torulopsis sp.                                                                          Methanol                                                                              4.5    4.9   1.0    0.21                                    Strain A.sub.1 (NRRLY -                                                       11,419)   Ethanol 4.2    4.7   1.2    0.20                                              Methylamine                                                                           4.3    4.5   0.9    0.12                                              Methylformate                                                                         4.5    4.9   1.1    0.25                                              Propylamine                                                                           3.2    3.8   0.62   0.10                                    Kloeckera sp.                                                                           Methanol                                                                              4.8    5.9   1.2    0.25                                    Strain A.sub.2 (NRRLY -                                                       11,420)   Ethanol 4.5    5.7   1.0    0.12                                              Methylamine                                                                           4.0    5.4   1.0    0.10                                              Methylformate                                                                         4.9    5.9   1.2    0.28                                              Propylamine                                                                           4.0    4.2   0.92   0.11                                    __________________________________________________________________________     .sup.a The products of oxidation were identified by gas chromatography        retention time comparison and cochromatography with authentic standard.       Analysis also revealed that no further oxidation of products                  (methylketones) occurred.                                                

Cell suspensions (2 g. wet weight) of packed cells in 10 ml. of 50 mMsodium phosphate buffer, pH 7.0 at 4° C. were disrupted by sonicationwith a Megason ultrasonic disintegration. The sonicated cell suspensionswere centrifuged for 15 minutes at 30,000×g. The supernatant liquid wastermed the crude extracts.

Purification of Secondary Alcohol Dehydrogenase Derived from Yeast

Large scale cultures of Pichia sp. NRRL Y-11,328 were grown withaeration at 30° C. in a 14-liter New Brunswick f fermentor in a mineralsalt medium containing methanol (0.4%, v/v) as the sole carbon source.The cells (200 g., wet weight) were suspended in 50 mm sodium phosphatebuffer, pH 7.0, containing 1 mM dithiothreitol (buffer A), and crudeextracts were prepared as described previously. To the crude extracts,18 ml. of protamine sulfate solution [2% solution in 0.1 M tris(hydroxymethyl) aminomethane (tris) base] was added dropwise withcontinuous stirring. After standing for 30 min., the extracts werecentrifuged at 20,000×g. for 60 min. The supernatant solution wasfractionated with solid ammonium sulfate. Extracts were brought to 50%of saturation with respect to ammonium sulfate by addition of 313 g. ofthe salt per liter of extract. Precipitated proteins was removed bycentrifugation, and 137 g. of ammonium sulfate was added per liter ofthe supernatant liquid to bring it to 70% of saturation. Materialprecipitating between 50 and 70% of saturation was collected bycentrifugation and dissolved in buffer A. This preparation was dialyzedovernight against buffer A, and the dialyzed material was applied to aDEAE-cellulose column (5×40 cm) that had been equilibrated with bufferA. The sample was washed with 200 ml. of buffer A and eluted with 2liters of buffer A that contained NaCl in a linear gradient running froma concentration of 0 to 0.5 M. Fractions of 15 ml. were collected.Fractions containing secondary alcohol dehydrogenase activity werepooled and were termed DEAE-cellulose eluate. The DEAE-cellulose eluatewas concentrated by ammonium sulfate fractionation. Materialprecipitating between 50 and 70% of ammonium sulfate saturation wascollected by centrifugation and dissolved in buffer A. This preparationwas dialyzed overnight against buffer A, and 4 ml. samples were passedthrough a Bio-Gel agarose A-1.5 column (2.5×100 cm) that had beenequilibrated with buffer A. Fractions containing constant specificactivity of enzyme were pooled and concentrated by Amiconultrafiltration using an XM 50 filter.

The reaction mixture, in a total of 3.0 ml., contained 50 mM phosphatebuffer, pH 7.0, 20μ mole NAD⁺, cell extracts (1 ml.). The reactions werestarted by the addition of 50 moles of secondary alcohol (isopropanol,2-butanol, 2-pentanol, 2-hexanol) and the rate of production of methylketones (acetone, 2-butanone, 2-pentanone, 2-hexanone) was measured bygas chromatography.

The ketone product obtained from oxidation of secalcohols by cellextracts of organisms were estimated by flame ionization gaschromatography by using a stainless steel column (12 ft. by 1/8 in.)packed with 10% Carbowax 20 M on 80/100 chromosorb w column (PerkinElmer Corp., Norwalk, Conn.). The column temperature was maintainedisothermally at 130° C. and the carrier gas flow was 30 ml. of heliumper min. The various ketone products (acetone, 2-butanone, 2-pentanone,2-hexanone) were identified by retention time comparisons andco-chromatography with authentic standard. The protein content ofcell-suspensions was determined by the Lowry et al. method.

Secondary alcohol dehydrogenase activity was measuredspectrophotometrically at 340 nm with a NAD⁺ as an electron acceptor.The reaction mixture, in a total 3.0 ml., contained 50 mM phosphatebuffer, pH 8.0, 5μ moles NAD⁺, crude extracts, and subtrate. Thereactions were started by addition of 100 μl of 0.1 M substrate and therate of NAD⁺ reduction was measured. Protein concentration wasdetermined by the method of Lowry et al.

Cell free extracts derived from yeasts, Candida utilis ATCC 26,387,Hansenula polymorpha ATCC 26,012, Pichia sp. NRRL Y-11,328, Torulopsissp. strain A₁ NRRL Y-11,419 and Kloeckera sp. strain A₂ NRRL Y-11,420grown on methanol catalyzed an NAD⁺ -dependent oxidation of secondaryalcohols (isopropanol, 2-butanol, 2-pentanol, 2-hexanol) to thecorresponding methyl ketones (acetone, 2-butanone, 2-pentanone,2-hexanone). The rate of production of methyl ketones from secondaryalcohols are shown in Table XXII. Oxidation of secondary alcohols werealso estimated spectrophotometrically by measuring the reduction ofNAD⁺. The specific activities (nmoles NAD⁺ reduced per min. per mg.protein) of 78, 85, 105, 62, and 90 were obtained with extracts derivedfrom Candida utilis ATCC 26,387, Hansenula polymorpha ATCC 26,012,Pichia sp. NRRL Y-11,328 Torulopsis sp. NRRL Y-11,419 strain A₁ andKloeckera sp. strain A₂ NRRL Y-11,420, respectively, using 2-butanol asa substrate.

                                      TABLE XXII                                  __________________________________________________________________________    OXIDATION OF SECONDARY ALCOHOLS TO METHYLKETONE                               BY CELL EXTRACTS OF YEASTS                                                                 Conversion Rate.sup.a                                                         μmoles/hr/mg Protein                                                       Isopropanol                                                                          2-Butanol to                                                                         2-Pentanol to                                                                        2-Hexanol to                                Organisms    to Acetone                                                                           2-Butanone                                                                           2-Pentanone                                                                          2-Hexanone                                  __________________________________________________________________________    Candida utilis                                                                ATCC 26,387  4.5    4.92   0.82   0.45                                        Hansenula polymorpha                                                          ATCC 26,012  4.8    5.2    1.0    0.51                                        Pichia sp.                                                                    NRRL-Y-11,328                                                                              5.5    6.2    1.2    0.60                                        Torulopsis sp.                                                                strain A.sub.1 NRRL-Y-11,419                                                               4.5    4.9    1.0    0.21                                        Kloeckera sp.                                                                 strain A.sub.2 NRRL-Y-11,420                                                               4.8    5.9    1.2    0.25                                        __________________________________________________________________________     .sup.a Reactions were carried out as described above. The products of         oxidation of secondary alcohols were identified and estimated by gas          chromatography.                                                          

The SADH enzyme was eluted from a DEAE-cellulose column at 0.08 M NaClconcentration. The overall 60-fold purification was achieved from crudeextracts. Purity of the enzyme preparation was examined bypolyacrylamide gel electrophoresis. The purified enzyme preparationsmigrated as a single protein band when subjected to electrophoresis onpolyacrylamide gel. Table XXIII illustrates a summary of thepurification steps and an analysis of the products at the end of eachstep.

The substrate specificity of the purified secondary alcoholdehydrogenase was examined spectrophotometrically. Among varioussecondary alcohls tested, the enzyme catalyzed the oxidation ofisopropanol, 2-butanol, 2-pentanol, and 2-hexanol.

2-Heptanol, 2-octanol, methanol, ethanol, propan-1-ol, butan-1-ol,pentan-1-ol, 1,2-propandiol, 1,2-butandiol and 1,3-butandiol were notoxidized by the purified enzyme.

The purified enzyme required NAD⁺ as an electron acceptor. NADP,phenazine methosulfate, potassium ferricyanide, cytochrome c,2,6-dichlorophenol indophenol, flavin adenine dinucleotide could not actas electron carrier.

Various primary alcohols not oxidized by secondary alcohol dehydrogenasewere tested as potential inhibitors of enzyme activity. Enzyme activitywas not inhibited by primary alcohols when tested at 10⁻³ M. Amongvarious sulfhydryl inhibitors and metal-binding compounds tested,p-hydroxy mercaribenzoate, glutathione, imidazole and 1,10phenanthroline were strongly inhibited secondary alcohol dehydrogenaseactivity. Enzyme activity was also inhibited by heavy metals such assilver nitrate, mercuric thiocyanate and cupric sulfate.

                  TABLE XXIII                                                     ______________________________________                                        PURIFICATION OF SECONDARY ALCOHOL                                             DEHYDROGENASE FROM Pichia sp. NRRL Y-11,328.sup.a                                                              Sp. activity                                            Vol.   Protein        (Units/mg.                                                                            Yield                                Step       (ml)   (mg)    Units  of Protein)                                                                           %                                    ______________________________________                                        Crude extracts                                                                           875    21,875  2391375                                                                              109     100                                  2. Protamine                                                                             890    21,360  2370960                                                                              111     99                                   sulfate treatment                                                             3. Ammonium sul-                                                                         117     3,090  1820010                                                                              589     76                                   fate fractionation                                                            (50-70%                                                                       saturation)                                                                   4. DEAE-cellulose                                                                         55     200     706800                                                                              3534    29                                   eluate                                                                        5. Bio-Gel  19      52     312624                                                                              6012    13                                   chromatography                                                                ______________________________________                                         .sup.a Secondary alcohol dehydrogenase activity was estimated                 spectrophotometrically as described above using 2butanol as a substrate.      Specific activity was expressed as nanomoles of NAD+ reduced per min per      mg of protein.                                                           

Acetone and 2-butanone was detected as the product of oxidation ofisopropanol and 2-butanol, respectively, by the purified enzyme. Theamount of NAD⁺ re uced and product formed is consistent withquantitative oxidation of both substrates. These results are shown inTable XXIV.

                  TABLE XXIV                                                      ______________________________________                                        STOICHIOMETRY OF ISOPROPANOL AND                                              SEC-BUTANOL OXIDATION BY THE PURIFIED                                         SECONDARY ALCOHOL DEHYDROGENASE                                               Substrate   NAD.sup.+ Reduced.sup.a                                                                     Product formed.sup.b                                (μmoles) (μmoles)   (μmoles)                                         ______________________________________                                        Isopropanol                                                                             5.7   5.4           Acetone   5.5                                   2-Butanol 6.0   5.9           2-Butanone                                                                              5.7                                   ______________________________________                                         .sup.a The estimation of NAD.sup.+ reduced was measured                       spectrophotometrically at 340 nm.                                             .sup.b The estimation of products was detected by gas chromatography as       described in the methods.                                                

ALKANE OXIDATION SYSTEM

Both cell suspensions (particulate fraction) and cell-free particulatefraction of methane-grown methylotroph microorganisms are capable ofcatalyzing the conversion of C₃ -C₆ n-alkanes to the correspondingalcohols including secondary alcohols. The conditions for preparing thecell suspensions or the cell-free particulate fractions frommethane-grown methylotroph microorganisms is the same as describedabove. The cell-free particulate fraction requires the presence ofoxygen and NADH as an electron donor. The conversion to the alcohol wasinhibited by metal-binding agents which suggests the involvement ofmetal ion(s) in the conversion of the alkanes to secondary alcohols.Propylene was also found to inhibit the conversion which suggests thatthe propylene and n-alkane (e.g., propane) are competing for the sameenzyme system(s). Ascorbate and reduced nicotinamide adeninedinucleotide phosphate (NADPH) could also be utilized as an electrondonor in place of NADH for the conversion. Tables XXV and XXVI show theconversion of C₃ -C₆ n-alkanes to the corresponding secondary alcoholsusing cell suspensions and cell-free particulate fractions,respectively, of methane-grown methylotroph microorganisms.

                                      TABLE XXV                                   __________________________________________________________________________    CONVERSION OF N-ALKANES TO SECONDARY ALCOHOLS                                 BY MICROORGANISMS.sup.a                                                                          Conversion Rate                                                               μmoles/hr/.sup.5 mg. protein                                               n-propane                                                                            n-butane                                                                           n-pentane                                                                           n-hexane                                               Growth                                                                             to     to   to    to                                       Microorganisms                                                                              Substrate                                                                          2-propanol                                                                           2-butanol                                                                          2-pentanol                                                                          2-hexanol                                __________________________________________________________________________    Methylosinus trichosporium                                                    (OB3b, NRRL-B-11,196)                                                                       Methane                                                                            2.5    1.5  0.06  0.01                                     Methylococcus capsulatus                                                      (Texas, ATCC 19,069                                                                         Methane                                                                            1.1    1.0   0.032                                                                              0.01                                     Methylobacter capsulatus                                                      (Y, NRRL-B-11,201)                                                                          Methane                                                                            0.20   0.09 --    --                                       Methylosinus sp.                                                              (CRL-15, NRRL-B-11,202)                                                                     Methane                                                                            2.1    1.2  --    --                                       Methylobacterium sp.                                                          (CRL-26, NRRL-B-11,208)                                                                     Methane                                                                            1.4    0.80 0.01   0.007                                   Methylomonas sp.                                                              (CRL-17, NRRL-B-11,208)                                                                     Methane                                                                            1.6    1.2  --    --                                       __________________________________________________________________________     .sup.a The product secondary alcohols were identified and estimated by GC     retention time comparison and cochromatography with authentic standards. 

                  TABLE XXVI                                                      ______________________________________                                        HYDROXYLATION OF N-ALKANES TO SECONDARY                                       ALCOHOLS BY PARTICULATE P(40).sup.a FRACTION                                  OF METHYLOTROPHS:                                                                             Conversion Rate                                                               μmoles/hr/2.0mg. of protein                                                  n-propane    n-butane                                                         to           to                                             Organisms         2-propanol   2-butanol                                      ______________________________________                                         Methylosinus sp.                                                             (CRL-15, NRRL-B-11,202)                                                                         1.5          0.89                                           Methylococcus capsulatus                                                                        1.2          0.92                                           (Texas, ATCC 19,069)                                                          Methylosinus trichosporium                                                                      1.32         0.79                                           (OB3b, NRRL-B-11,196)                                                         Methylobacterium sp.                                                          (CRL-26, NRRL-B-11,222)                                                                         1.0          0.61                                           ______________________________________                                         .sup.a Particulate P(40) fraction was prepared as follows: Cellsuspension     at 4° C. were disintegrated through a French Pressure cell and         centrifuged at 4000 × g. for 15 min. to remove unbroken bacteria.       The supernatant solution was then centrifuged at 40,000 × g. for 30     min. at 4° C., yielding the particulate P(40) and soluble S(40)        fractions. The products were identified by gas chromatography and             cochromatography with authentic standard.                                

Table XXVII shows that cell suspensions of methane-grown methylotrophmicroorganisms convert C₁ -C₂ alkanes to the corresponding alcohols andpropane and butane are converted to a plurality of oxidation products,including primary and secondary alcohols, methyl ketones and aldehydes.

                  TABLE XXVII                                                     ______________________________________                                        CONVERSION OF n-ALKANES                                                       TO OXIDATION PRODUCTS.sup.a                                                              Conversion Rate                                                               μmoles/hr./mg./protein                                                                          Methylococcus                                                  Methylosinus   capsulatus                                                     trichosporium  CRL M1 NRRL                                   Substrate                                                                            Products  OB3b NRRL B-11,196                                                                           B-11,219                                      ______________________________________                                        Methane                                                                              Methanol  1.5            2.5                                           Ethane Ethanol   1.3            2.0                                           Propane                                                                              1-Propanol                                                                              0.4            0.5                                           Propane                                                                              2-Propanol                                                                              0.6            0.7                                           Propane                                                                              Propanol  0.1            0.2                                           Propane                                                                              Acetone   0.2            0.3                                           Butane 1-Butanol 0.3            0.4                                           Butane 2-Butanol 0.4            0.5                                           Butane 2-Butanone                                                                              0.1            0.2                                           Butane n-butanol 0.1            0.2                                           ______________________________________                                         .sup.a Cell-suspensions of methanegrown methylotroph microorganisms           indicated in 0.15 M phosphate buffer, pH 7.0 incubated in the alkanes as      indicated at 3° C. The oxidation products were determined by g.l.c                                                                              

Leadbetter and Foster (Archiv. fur Mikrobiologie, 35:92-104 (1960))reported that methane grown Pseudomonas methanica co-oxidized propaneand butane to their corresponding methyl ketones. They stated thatresting cell-suspensions of methane-grown cells, however, did notoxidize propane or butane. Later, Lukins and Foster (J. Bacteriol.,85:1074-1086 (1963)) reported that propane-grown Mycobacterium smegmatis422 oxidized n-alkanes to their corresponding methyl ketones. We havefound and demonstrated that resting cell-suspensions of methane-growncells oxidize C₃ -C₆ alkanes to their corresponding secondary alcoholsand methyl ketones in the absence of growth substrates. In addition, wehave demonstrated for the first time the conversion of C₃ -C₆ secondaryalcohols to their corresponding methyl ketones by resting cellsuspensions (particulate fraction) of either alkane-grown or alcoholgrown cells. Succinate-grown cells do not have SADH activity, suggestingthat either alkane or alcohol is required for inducing the enzyme.

As shown above, cell suspensions of these new cultures as well as knownC₁ -utilizers grown on either methane or methanol oxidized secondaryalcohols to their corresponding methyl ketones. The cultures tested wereselected from distinct general and they were compared for their optimalconditions in the production of 2-butanone. These cultures were:Methylosinus trichosporium OB3b (NRRL B-11,196) (a Type I obligatemethane-utilizer); Methylobacterium organophilum CRL 26 (NRRL B-11,222)(a facultative methane-utilizer); Hansenula polymorpha ATCC 26012; andPseudomonas sp. ATCC 21439 (an obligate methanol-utilizer). The rate of2-butanone production was linear for the first 4 hours of incubation forall five cultures tested. The yeast culture had the highest productionrate. The optimum temperature for the production of 2-butanone was 35°C. for all the bacteria tested. The yeast culture had a highertemperature optimum (40° C.), and a reasonably high 2-butanoneproduction rate was also observed at 45° C. for this yeast. Theproduction of 2-butanone was affected by substrate concentration andcell concentration. The inhibition by metal-chelating agents of theproduction of 2-butanone suggests the involvement of metal(s). Noproduct (2-butanone) inhibition was observed in any of thecell-suspensions from all the five cultures tested.

We have found that cell-free soluble extracts from sonically disruptedcells also oxidize 2-butanol to 2-butanone. The cell-free systemrequires addition of a cofactor, specifically NAD, for its activity. Oneof the explanations for the rate decreases in 2-butanone productionafter 4 hours of incubation, therefore, may be the depletion of NAD inthe cell suspensions.

Nicotinamide adenine dinucleotide (NAD) was found to be a requirementfor the oxidation of C₃ -C₆ secondary alcohols in the cell-free SADHsystem. Other cofactors tested (including PMS, GSH, FAD, potassiumferricyanide, dichlorophenol indophenol, and NADP) were not effective.

The molecular weight of the pure SADH as estimated by a Bio-Gel agaroseA-1.5 column is 95,000 dalton. Acrylamide gel electrophoresis of thepurified SADH fraction from the affinity chromatography showed a singleprotein band. The Km values for 2-butanol and NAD are 0.25 mM and 0.011mM, respectively. The pH optimum for SADH activity was around 8-9 (0.05M sodium phosphate buffer for pH 5 to 8; 0.05 M sodium pyrophosphatebuffer for pH 8 to 11).

SADH oxidizes C₃ -C₆ secondary alcohols with the following relativepercent rate: 2-propanol (85%), 2-butanol (100%), 2-pentanol (5%),2-hexanol (2%), acetaldehyde (4%), propanol (2%), cyclohexanol (4%),butane 1,3-diol (2%), and butane 2,3-diol (2.5%). The followingcompounds tested were not oxidized by SADH: 2-heptanol to 2-decanol,formaldehyde, butanal to decanal, benzaldehyde, methanol to n-decanol,isobutanol, phenol, butane 1,2-diol, and succinic acid. It seems that ahydrophobic carbon moiety adjacent to the secondary alcohol is requiredfor the enzyme activity.

The SADH activity was inhibited by metal-chelating agents in thefollowing order (percent inhibition): 1,10-phenanthroline (95%),α,α-bipyridyl (70%), EDTA (63%), and sodium azide (10%). This suggestspossible metal involvement. However, the activity was not inhibited bysodium cyanide or thiourea. The enzyme activity was also inhibited bystrong thio inhibitors such as ρ-hydroxy mercuribenzoate (100%) and5,5'-dithiobis (2-nitrobenzoic acid) and was not inhibited by lesspotent thio inhibitors such as iodoacetic acid or N-ethylmaleimide. Thephysiological significance of this SADH in methylotrophs as well asother gaseous hydrocarbon utilizers is not known. However, possessingthis enzyme is of great advantage to the organism as its growth yield,when growing on gaseous alkanes as the sole source of carbon and energy,could be exclusively NAD(P)H-dependent. Secondary alcohols areintermediates in the oxidation of n-alkanes by either Pseudomonas orMycobacterium. The methane monooxygenase from Methylococcus capsulatus(Bath) also oxidizes n-alkanes to both primary and secondary alcohols.The fact that SADH is also present in the methanol-grown cells indicatesthat the enzyme is not induced by n-alkanes.

The metabolism of the obligate methylotrophs is uniquely dependent on aone-carbon compound (formaldehyde) for the biosynthesis of certainessential cellular constituents. This compound can be obtained frommethane and methanol, but is unobtainable from the non-growth-supportingcompounds.

NAD-dependent alcohol dehydrogenase and PMS-dependent methanoldehydrogenase are well characterized enzymes. Both of thesedehydrogenases have a broad specificity toward primary alcohols.Recently, Metha (J. Bacteriol., 124:1165-1167 (1975)) reported anNAD-linked alcohol dehydrogenase from a yeast grown on methanol. Thisprimary alcohol dehydrogenase also oxidizes 2-propanol. In addition, thereport stated that this alcohol dehydrogenase was very unstable that itlost all of its enzyme activity within 24 hours after fourfoldpurification. Results from our preliminary studies, however, indicatethat our secondary alcohol dehydrogenase is a secondary alcohol-specificenzyme with highest activity on 2-propanol and 2-butanol, and has noactivity towards primary alcohols.

What is claimed is:
 1. A process for converting a C₃ -C₆ secondaryalcohol to the corresponding methyl ketone comprising oxidizing saidalcohol by contacting said alcohol in a medium, under aerobicconditions, with microbial cells derived from a methylotrophicmicroorganism or an enzyme preparation derived from said cells, whereinsaid microorganism has been previously grown under aerobic conditions ina nutrient medium containing a carbon-containing compound which providesthe carbon and energy source for growth of the cells and induces C₃ -C₆secondary alcohol dehydrogenase enzyme activity in said cells, andwherein said microorganism is selected from the group consisting of:

    ______________________________________                                        Methylosinus trichosporium                                                                    (CRL 15 PM1)                                                                              NRRL B-11,202;                                    Methylosinus sporium                                                                          (CRL 16 PM2)                                                                              NRRL B-11,203;                                    Methylocystis parvus                                                                          (CRL 18 PM4)                                                                              NRRL B-11,204;                                    Methylomonas methanica                                                                        (CRL M4P)   NRRL B-11,205;                                    Methylomonas methanica                                                                        (CRL 21 PM7)                                                                              NRRL B-11,206;                                    Methylomonas albus                                                                            (CRL M8Y)   NRRL B-11,207;                                    Methylomonas                                                                  streptobacterium                                                                              (CRL 17 PM3)                                                                              NRRL B-11,208;                                    Methylomonas agile                                                                            (CRL 22 PM9)                                                                              NRRL B-11,209;                                    Methylomonas rubrum                                                                           (CRL M6P)   NRRL B-11,210;                                    Methylomonas rubrum                                                                           (CRL 20 PM6)                                                                              NRRL B-11,211;                                    Methylomonas rosaceus                                                                         (CRL M10P)  NRRL B-11,212;                                    Methylomonas rosaceus                                                                         (CRL M7P)   NRRL B-11,213;                                    Methylobacter chroococcum                                                                     (CRL M6)    NRRL B-11,214;                                    Methylobacter chroococcum                                                                     (CRL 23 PM8)                                                                              NRRL B-11,215;                                    Methylobacter bovis                                                                           (CRL M1Y)   NRRL B-11,216;                                    Methylobacter bovis                                                                           (CRL 19 PM5)                                                                              NRRL B-11,217;                                    Methylobacter vinelandii                                                                      (CRL M5Y)   NRRL B-11,218;                                    Methylococcus capsulatus                                                                      (CRL M1)    NRRL B-11,219;                                    Methylococcus minimus                                                                         (CRL 24 PM12)                                                                             NRRL B-11,220;                                    Methylococcus capsulatus                                                                      (CRL 25 PM13)                                                                             NRRL B-11,221;                                    Methylobacterium                                                                              (CRL 26 R6) NRRL B-11,222;                                    organophilum                                                                  Pichia sp.      (CRL-72)    NRRL-Y-11,328;                                    Torulopsis sp.  (A.sub.1)   NRRL-Y-11,419;                                    Kloeckera sp.   (A.sub.2)   NRRL-Y-11,420;                                    ______________________________________                                    

and mutants thereof wherein said methyl ketone is produced in isolableamounts.
 2. The process of claim 1 wherein the enzyme preparation is abacterial or yeast cell suspension which has been grown on a methylradical donating compound.
 3. The process of claim 2 wherein said enzymepreparation comprises microbial cells.
 4. The process of claim 1 whereinsaid enzyme preparation is cell-free and the medium additionallycontains nicotinamide adenine dinucleotide.
 5. The process of claim 4wherein the enzyme preparation is a bacterial- or yeast-derivedcell-free extract.
 6. The process of claim 4 wherein the enzymepreparation is substantially purified C₃ -C₆ secondary alcoholdehydrogenase.
 7. The process of claim 4 wherein the conversion iscarried out at a temperature ranging from about 5° to about 55° C. andat a pH in the range from about 4 to about
 9. 8. The process of claim 4wherein the enzyme is immobilized.
 9. The process of claim 4 wherein theconversion is carried out batchwise.
 10. The process of claim 4 whereinthe conversion is carried out in a continuous manner and said enzyme isimmobilized.
 11. The process of claim 4 wherein the enzyme, C₃ -C₆secondary alcohol dehydrogenase, has a molecular weight of 95,000±3,000dalton as determined by Bio-gel column chromatography and two zinc atomsper molecule of protein.